Theoretical and Practical Approaches to Evaluate Suitable Primer Sets for the Analysis of Soil Fungal Communities

ABSTRACTUltra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predictedin silicoand by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.

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COMPARISON EFFICACY OF ITS AND 18S rDNA PRIMERS FOR DETECTION OF FUNGAL DIVERSITY IN COMPOST MATERIAL BY PCR-DGGE TECHNIQUE

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/15/4/13416 ◽

2018 ◽

Vol 15 (4) ◽

pp. 729-735

Author(s):

Pham Ngoc Tu Anh ◽

Pham Thi Thu Hang ◽

Le Thi Quynh Tram ◽

Nguyen Thanh Minh ◽

Dinh Hoang Dang Khoa

Keyword(s):

Fungal Diversity ◽

Pcr Amplification ◽

Fungal Communities ◽

Variable Regions ◽

Pcr Inhibitors ◽

Composting Process ◽

Pcr Products ◽

Pure Cultures ◽

Primer Sets ◽

Aspergillus Sp

Through composting process, biosolid wastes are gradually transformed into compost material which can be used as soil fertilizer. Among microorganisms involved in composting process, fungi play important roles because they break down complex substrates, such as ligno-cellulose. Recently, PCR-DGGE technique has been considered as a useful tool for analysis of fungal diversity in environmental samples. Among other factors, primer set selection is necessary for successful of the PCR-DGGE analysis. There are several PCR primer sets targeting fungal variable regions of 18S ribosomal DNA (rDNA) and internal transcribed spacer (ITS) for the use in community analyses, however there exist just few reports on efficacy of these primers in studying fungal communities in compost materials. In this study, four different primer sets were tested, including EF4/Fung5 (followed by EF4/NS2-GC), EF4/ITS4 (followed by ITS1F-GC/ITS2), NS1/GC-Fung, and FF390/FR1-GC. Extracted DNA from compost materials often contains co-extracted humic substances and other PCR inhibitors. Therefore, the primers were tested for (i) tolerance to the PCR inhibitors presenting in the DNA extracted from compost materials, and (ii) efficacy and specificity of the PCR. The results showed that of the four primer sets, only FF390/FR1-GC achieved both criteria tested whereas the other three did not, i.e. primer EF4/ITS4 had low tolerance to PCR inhibitors, primers EF4/Fung5 was low in PCR amplification efficacy, whereas primers EF4/ITS4 created unspecific products. DGGE analyses of PCR products amplified with the primer set FF390/FR1-GC showed single bands for reference pure cultures Penicillium sp., Aspergillus sp., and Trichoderma sp., as well as distinctly separated bands for the fungal communities of three different composting materials. Thus, the primer set FF390/FR1-GC could be suitable for studying structure and dynamic of fungal communities in compost materials.

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Novel ITS1 Fungal Primers for Characterization of the Mycobiome

mSphere ◽

10.1128/msphere.00488-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 21

Author(s):

Mykhaylo Usyk ◽

Christine P. Zolnik ◽

Hitesh Patel ◽

Michael H. Levi ◽

Robert D. Burk

Keyword(s):

Pcr Primers ◽

Fungal Communities ◽

Rrna Gene ◽

Its1 Region ◽

Sequencing Technologies ◽

Identification Of Fungi ◽

Fungal Organisms ◽

Primer Sets ◽

Yeasts And Molds

ABSTRACT The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states. Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states.

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Selection and Experimental Evaluation of Universal Primers to Study the Fungal Microbiome of Higher Plants

Phytobiomes Journal ◽

10.1094/pbiomes-02-18-0009-r ◽

2018 ◽

Vol 2 (4) ◽

pp. 225-236 ◽

Cited By ~ 2

Author(s):

Silvia Scibetta ◽

Leonardo Schena ◽

Ahmed Abdelfattah ◽

Sonia Pangallo ◽

Santa O. Cacciola

Keyword(s):

Fungal Diversity ◽

Higher Plants ◽

Fungal Communities ◽

Universal Primers ◽

Its2 Region ◽

Its1 Region ◽

Perfect Set ◽

Fungal Microbiome ◽

Primer Sets ◽

The Impact

The impact of primer choice on results of metabarcoding studies was experimentally evaluated by analyzing fungal communities associated with leaves of four plant species. Significant differences in target specificity of primers were highlighted by a percentage of plant reads ranging from almost nothing to 30 to 35% of the total detected sequences. Overall, primer sets targeting the internal transcribed spacer 1 (ITS1) region proved to be more specific than those targeting the ITS2 region. A comparable taxa coverage was revealed for all investigated primer sets. However, each primer set detected only around 50% of the overall detected taxa highlighting that a consistent part of the actual fungal diversity remains undetected in studies conducted using a single couple of primers. The coverage was increased to 70 to 80% by combining results from two different primer sets. Some fungal taxa were preferentially or exclusively detected by certain primer sets and this association between primers and taxa was generally recurrent on several plant hosts. Data highlighted that a perfect set of primers to investigate the whole fungal diversity does not exist and that whatever the choice, only a fraction of the actual microbial diversity will be investigated. However, provided information may be valuable to select the best primers according to the objective of the analysis.

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Investigation and analysis of microbiological communities in natural Ophiocordyceps sinensis

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0610 ◽

2015 ◽

Vol 61 (2) ◽

pp. 104-111 ◽

Cited By ~ 9

Author(s):

Fei Xia ◽

Yan Liu ◽

Guang-Rong Shen ◽

Lian-Xian Guo ◽

Xuan-Wei Zhou

Keyword(s):

Bacterial Flora ◽

Fungal Species ◽

Fungal Communities ◽

Rrna Gene ◽

Internal Transcribed Spacer Region ◽

Fungal Community Structure ◽

Ophiocordyceps Sinensis ◽

Medicinal Fungus ◽

Primer Sets ◽

Pcr Techniques

Ophiocordyceps sinensis is a fungus that parasitizes caterpillars, and more than 30 species of filamentous fungi have been isolated from its fruiting body. However, its microbiological diversity remains unclear. Based on the clone library and quantitative PCR techniques, the bacterial flora and mycobiota of 3 different samples (larva, stromata/sclerotia, and surface soil) from natural O. sinensis specimens were investigated using primer sets that targeted the 16S rRNA gene and internal transcribed spacer region of ribosomal DNA. The results showed that the abundance of bacterial and fungal communities in the soil attached to the surface of O. sinensis was (6.4 ± 1.4) × 106 and (6.0 ± 0.3) × 107 copies/g dry matter, respectively, which was the highest compared with that in the larva and stromal samples. The main groups of bacteria in the O. sinensis samples were Proteobacteria and Actinobacteria, while Ascomycota was the most dominant fungal group in the 3 samples. At the genus level, Geomyces, Phoma, and Trichocladium were the dominant genera in the larval sample, while Geomyces and Cladosporium were the dominant genera in the stromal sample. In conclusion, a great number of bacterial and fungal species were present in naturally occurring O. sinensis specimens, and there was a high diversity of bacterial and fungal communities. These findings contribute to the understanding of the bacterial and fungal community structure of this valuable medicinal fungus and lay the foundation for the future discovery of new medicinal microorganism resources.

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Comparision of two different primer sets for PCR detection of transfusion transmitted virus DNA in blood donors, hemodialysis patients and patients with thalassemia major, chronic HBV and HCV infection

Journal of Hepatology ◽

10.1016/s0168-8278(01)80596-1 ◽

2001 ◽

Vol 34 (0) ◽

pp. 163 ◽

Cited By ~ 1

Author(s):

H Verdi

Keyword(s):

Blood Donors ◽

Thalassemia Major ◽

Hemodialysis Patients ◽

Pcr Detection ◽

Hcv Infection ◽

Transmitted Virus ◽

Chronic Hbv ◽

Primer Sets ◽

Transfusion Transmitted Virus

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Experimentally altered rainfall regimes and host root traits affect grassland arbuscular mycorrhizal fungal communities

10.32942/osf.io/cu4jh ◽

2019 ◽

Author(s):

Coline Deveautour ◽

Suzanne Donn ◽

Sally Power ◽

Kirk Barnett ◽

Jeff Powell

Keyword(s):

Fungal Community ◽

Community Assembly ◽

Root Length ◽

Specific Root Length ◽

Root Traits ◽

Arbuscular Mycorrhizal ◽

Fungal Communities ◽

Rainfall Regime ◽

Precipitation Regimes ◽

Rainfall Regimes

Future climate scenarios predict changes in rainfall regimes. These changes are expected to affect plants via effects on the expression of root traits associated with water and nutrient uptake. Associated microorganisms may also respond to these new precipitation regimes, either directly in response to changes in the soil environment or indirectly in response to altered root trait expression. We characterised arbuscular mycorrhizal (AM) fungal communities in an Australian grassland exposed to experimentally altered rainfall regimes. We used Illumina sequencing to assess the responses of AM fungal communities associated with four plant species sampled in different watering treatments and evaluated the extent to which shifts were associated with changes in root traits. We observed that altered rainfall regimes affected the composition but not the richness of the AM fungal communities, and we found distinctive communities in the increased rainfall treatment. We found no evidence of altered rainfall regime effects via changes in host physiology because none of the studied traits were affected by changes in rainfall. However, specific root length was observed to correlate with AM fungal richness, while concentrations of phosphorus and calcium in root tissue and the proportion of root length allocated to fine roots were correlated to community composition. Our study provides evidence that climate change and its effects on rainfall may influence AM fungal community assembly, as do plant traits related to plant nutrition and water uptake. We did not find evidence that host responses to altered rainfall drive AM fungal community assembly in this grassland ecosystem.

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Temporal dynamics of mycorrhizal fungal communities and co-associations with grassland plant communities following experimental manipulation of rainfall

10.32942/osf.io/t82ug ◽

2019 ◽

Cited By ~ 1

Author(s):

Coline Deveautour ◽

Sally Power ◽

Kirk Barnett ◽

Raul Ochoa-Hueso ◽

Suzanne Donn ◽

...

Keyword(s):

Community Composition ◽

Plant Community ◽

Plant Communities ◽

Fungal Community ◽

Mycorrhizal Fungi ◽

Temporal Dynamics ◽

Arbuscular Mycorrhizal ◽

Fungal Communities ◽

Plant Responses ◽

Rainfall Regimes

Climate models project overall a reduction in rainfall amounts and shifts in the timing of rainfall events in mid-latitudes and sub-tropical dry regions, which threatens the productivity and diversity of grasslands. Arbuscular mycorrhizal fungi may help plants to cope with expected changes but may also be impacted by changing rainfall, either via the direct effects of low soil moisture on survival and function or indirectly via changes in the plant community. In an Australian mesic grassland (former pasture) system, we characterised plant and arbuscular mycorrhizal (AM) fungal communities every six months for nearly four years to two altered rainfall regimes: i) ambient, ii) rainfall reduced by 50% relative to ambient over the entire year and iii) total summer rainfall exclusion. Using Illumina sequencing, we assessed the response of AM fungal communities sampled from contrasting rainfall treatments and evaluated whether variation in AM fungal communities was associated with variation in plant community richness and composition. We found that rainfall reduction influenced the fungal communities, with the nature of the response depending on the type of manipulation, but that consistent results were only observed after more than two years of rainfall manipulation. We observed significant co-associations between plant and AM fungal communities on multiple dates. Predictive co-correspondence analyses indicated more support for the hypothesis that fungal community composition influenced plant community composition than vice versa. However, we found no evidence that altered rainfall regimes were leading to distinct co-associations between plants and AM fungi. Overall, our results provide evidence that grassland plant communities are intricately tied to variation in AM fungal communities. However, in this system, plant responses to climate change may not be directly related to impacts of altered rainfall regimes on AM fungal communities. Our study shows that AM fungal communities respond to changes in rainfall but that this effect was not immediate. The AM fungal community may influence the composition of the plant community. However, our results suggest that plant responses to altered rainfall regimes at our site may not be resulting via changes in the AM fungal communities.

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