18S rRNA gene variation among common airborne fungi,and development of specific oligonucleotide probes for the detection of fungal isolates

In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.     

18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.     

Development and optimization of an 18S rRNA‐based oligonucleotide microarray for the fungal order Eurotiales

Aims: For identification of members of the fungal order Eurotiales, an 18S rRNA gene‐based oligonucleotide microarray was developed and optimized. Methods and Results: Eurotiales‐specific probes covering most members of the Eurotiales as well as species‐specific probes were designed and evaluated with three pure cultures (two fungi from the Eurotiales and one fungus from the Hypocreales). Nearly complete 18S rRNA genes of each reference culture were amplified and fluorescently labelled by random priming. Conclusions: Positive and negative hybridization results confirmed that the Eurotiales probes tested in this study could correctly identify members of the Eurotiales. The species‐specific probes were also capable of species‐level detection. Significance and Impact of the Study: These findings demonstrate the potential applications of a phylogenetic oligonucleotide microarray approach to characterizing fungal species and populations in environmental and other samples.     

Design and evaluation of an oligonucleotide-microarray for the detection of different species of the genus Kitasatospora

An oligonucleotide-microarray method was developed for the detection of Kitasatospora species in soil samples. The 16S-23S rDNA internal transcribed spacer (ITS) sequence of these antibiotics-producing actinomycetes was applied to design short oligonucleotide probes. Two different 26-mers were synthesized, specific to each species used. Additionally, four oligonucleotide probes were designed to evaluate the system. The oligonucleotides were spotted onto slides of the ArrayTube microarray system and examined with a new silver-labeling detection technique. Prior to hybridization analysis, the 16S-23S rDNA were amplified by polymerase chain reaction both from bacterial cells and environmental samples using two actinomycetes specific primers containing a 5' biotin labeling. The type strains of eight Kitasatospora species included in this study were K. phosalacinea DSM 43860, K. setae DSM 43861, K. cochleata DSM 41652, K. cystarginea DSM 41680, K. azatica DSM 41650, K. mediocidica DSM 43929, K. paracochleata DSM 41656, and K. griseola DSM 43859. The actinomycetes-specific primers were shown to amplify the entire 16S-23S rDNA ITS region from all tested strains. More importantly, the described technique allows the detection of Kitasatospora strains from soil samples by extracting metagenomic DNA followed by a PCR amplification step. This indicates that the oligonucleotide-microarray method developed in this study is a reliable tool for the detection of Kitasatospora species in environmental samples.     

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.     

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.     

A protocol for PCR in situ hybridization of hyphomycetes.

A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for the detection of hyphomycetes is presented. The experiments are exemplary carried out with strains of the genera Penicillium and Cladosporium. The small ribosomal subunit is amplified in situ by PCR using fungal specific primers. The amplicon is used as target region for a fluorescein-marked probe. The permeability of the fungal cell wall for the primers and the probe can be successfully achieved by enzymatic treatment with beta-glucanase. The protocol can be used as a basis for further development of in situ hybridization with taxon specific probes.     

A novel PCR-based approach for the detection and classification of potential cellulolytic fungal strains isolated from museum items and surrounding indoor environment

Aims: To develop a novel PCR‐based method able to detect potential cellulolytic filamentous fungi and to classify them exploiting the amplification of the cellobiohydrolase gene (cbh‐I) and its polymorphism. Methods and Results: A mixed approach including the combination of (i) fungal cultivation and isolation, (ii) classification of fungal isolates through the amplification of the cbh gene using a fluorescently labelled primer (f‐CBH‐PCR) and (iii) final fungal identification based on amplification and sequencing of the ITS1‐5.8S rDNA‐ITS2 region of the selected fungal strains was developed. By this approach, it was possible to screen 77 fungal strains belonging to 14 genera and 26 species. Conclusions: The f‐CBH‐PCR permitted the discrimination of fungal species, producing typical f‐CBH profiles. Significance and Impact of the Study: In this study, the cbh gene was used as a preliminary classification tool able to differentiate among themselves the fungal members isolated from indoor museum items and surrounding environment. Such mixed approach consented the fast identification of all isolated fungal strains. The f‐CBH‐PCR method demonstrated its discrimination power, and it can be considered as a new molecular system suitable for the classification of fungal strains isolated from different environments.     

Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys.     

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.     

Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH)

In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period.     

Specific detection of Arcobacter and Campylobacter strains in water and sewage by PCR and fluorescent in situ hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10(3) cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.     

Isolation of a specific DNA fragment and development of a PCR-based method for the detection of Mycobacterium genavense

The rise of Mycobacterium genavense infections is making identification ever more important for diagnosis and treatment. Moreover, isolation and identification of M. genavense are made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplification by PCR or similar techniques represents the only possibility of detecting and identifying M. genavense from tissue samples. In order to set up a simple and species-specific method based on the use of PCR and non-radioactive hybridization technique, we decided to search for and clone a specific DNA fragment of this bacterial species. In the present study, a 1734-bp fragment was isolated. This fragment was found to be highly specific for M. genavense strains. A species-specific pair of primers (MG22 and MG23) and two oligonucleotide probes (MG18 and MG19) were selected. They were successfully used to amplify and detect a 155-bp DNA fragment from the 13 available strains of M. genavense which were isolated from clinical specimens or from birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, although it is closely related to M. genavense. The present PCR technique uses species-specific primers for M. genavense. Followed by a non-radioactive hybridization technique on microplates it is able to distinguish M. genavense from other mycobacteria in one step, without sequencing or restriction analysis. On the basis of the Southern blot hybridization, PCR and sandwich hybridization results, we concluded that the isolated 1.7-kb sequence was specific for the M. genavense chromosome. The method developed here for M. genavense identification uses a simple methodology and commonly available reagents. Furthermore it can be easily automated.     

Efficient genetic analysis of fungal samples

We present a method for rapid genetic analysis of small amounts of fungal material. Sterile glass slides, sufficiently small to fit in a standard PCR tube, were placed on agar inside a Petri dish. After a few days, fungal cultures start to overgrow the glass slides. Glass slides with attached mycelium were harvested, analysed microscopically, and placed into a standard PCR tube. Conserved primers flanking the ITS regions of rDNA repeat were used in a direct PCR with the fungal material. Sequence data were generated to be included in phylogenetic analyses to investigate the relationships of the studied mycorrhizal fungi from orchids. The mycelium attached to glass slides was also used for an in situ hybridization experiment using fluorescent labelled oligonucleotides as probes. Fluorescent signal was found throughout the cytoplasm when a probe specific to a site in the nuclear small subunit rRNA is used.     

Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10³ cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.     

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.     

Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (approximately 14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.     

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

AIMS: To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. METHODS AND RESULTS: Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. CONCLUSIONS: Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates.