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

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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Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

Applied and Environmental Microbiology ◽

10.1128/aem.00905-17 ◽

2017 ◽

Vol 83 (17) ◽

Cited By ~ 40

Author(s):

Francesca De Filippis ◽

Manolo Laiola ◽

Giuseppe Blaiotta ◽

Danilo Ercolini

Keyword(s):

Microbial Ecology ◽

High Throughput ◽

Biological Samples ◽

Fungal Diversity ◽

High Throughput Sequencing ◽

Amplicon Sequencing ◽

Fungal Species ◽

Fungal Communities ◽

Its2 Region ◽

Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

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Impact of Bactrocera oleae on the fungal microbiota of ripe olive drupes

10.1101/342543 ◽

2018 ◽

Author(s):

Ahmed Abdelfattah ◽

David Ruano-Rosa ◽

Santa O. Cacciola ◽

Maria G. Li Destri Nicosia ◽

Leonardo Schena

Keyword(s):

Fruit Fly ◽

Fungal Communities ◽

Bactrocera Oleae ◽

Olive Fruit Fly ◽

Slight Variation ◽

Olive Fruit ◽

Sampling Locations ◽

Total Dna ◽

Fungal Microbiome ◽

The Impact

AbstractThe olive fruit fly (OFF), Bactrocera oleae is the most devastating pest affecting olive fruit worldwide. Previous investigations have addressed the fungal microbiome associated with olive drupes or B. oleae, but the impact of the insect on fungal communities of olive fruit remains undescribed. In the present work, the fungal microbiome of olive drupes, infested and non-infested by the OFF, was investigated in four different localities and cultivars. Olive fruit fly infestations caused a general reduction of the fungal diversity, a higher quantity of the total DNA and an increase in taxa that remained unidentified or had unknown roles. The infestations led to imbalanced fungal communities with the growth of taxa that are usually outcompeted. While it was difficult to establish a cause-effect link between fly infestation and specific fungi, it is clear that the fly alters the natural microbial balance, especially the low abundant taxa. On the other hand, the most abundant ones, were not significantly influenced by the insect. In fact, despite the slight variation between the sampling locations, Aureobasidium, Cladosporium, and Alternaria, were the dominant genera, suggesting the existence of a typical olive fungal microbiome.

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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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Responses of Soil Fungal Communities to Lime Application in Wheat Fields in the Pacific Northwest

Frontiers in Microbiology ◽

10.3389/fmicb.2021.576763 ◽

2021 ◽

Vol 12 ◽

Author(s):

Chuntao Yin ◽

Daniel C. Schlatter ◽

Duncan R. Kroese ◽

Timothy C. Paulitz ◽

Christina H. Hagerty

Keyword(s):

Pacific Northwest ◽

Fungal Community ◽

Fungal Diversity ◽

Soil Depth ◽

Fungal Communities ◽

Agricultural Practice ◽

The Pacific ◽

Soil Fungal Community ◽

Lime Application ◽

The Impact

Liming is an effective agricultural practice and is broadly used to ameliorate soil acidification in agricultural ecosystems. Our understanding of the impacts of lime application on the soil fungal community is scarce. In this study, we explored the responses of fungal communities to liming at two locations with decreasing soil pH in Oregon in the Pacific Northwest using high-throughput sequencing (Illumina MiSeq). Our results revealed that the location and liming did not significantly affect soil fungal diversity and richness, and the impact of soil depth on fungal diversity varied among locations. In contrast, location and soil depth had a strong effect on the structure and composition of soil fungal communities, whereas the impact of liming was much smaller, and location- and depth-dependent. Interestingly, families Lasiosphaeriaceae, Piskurozymaceae, and Sordariaceae predominated in the surface soil (0–7.5 cm) and were positively correlated with soil OM and aluminum, and negatively correlated with pH. The family Kickxellaceae which predominated in deeper soil (15–22.5 cm), had an opposite response to soil OM. Furthermore, some taxa in Ascomycota, such as Hypocreales, Peziza and Penicillium, were increased by liming at one of the locations (Moro). In conclusion, these findings suggest that fungal community structure and composition rather than fungal diversity responded to location, soil depth and liming. Compared to liming, location and depth had a stronger effect on the soil fungal community, but some specific fungal taxa shifted with lime application.

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Degenerate ITS7 primer enhances oomycete community coverage and PCR sensitivity to Aphanomyces species, economically important plant pathogens

Canadian Journal of Microbiology ◽

10.1139/cjm-2017-0100 ◽

2017 ◽

Vol 63 (9) ◽

pp. 769-779 ◽

Cited By ~ 5

Author(s):

A. Esmaeili Taheri ◽

S. Chatterton ◽

B.D. Gossen ◽

D.L. McLaren

Keyword(s):

Plant Pathogens ◽

Perfect Match ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Abundant Species ◽

Aphanomyces Euteiches ◽

Its1 Region ◽

Accurate Picture ◽

Primer Sets ◽

The Impact

Metagenomic analysis of oomycetes through deep amplicon sequencing has been conducted primarily using the ITS6–ITS7 primer set that targets the ITS1 region. While this primer set shows a perfect match to most oomycete taxa, ITS7 contains 3 mismatches to the corresponding binding site of plant pathogens within the genus Aphanomyces. Polymerase chain reaction (PCR) efficiency differs for taxa with uneven primer matching characteristics, which may explain why previous studies have detected this genus at low abundance. To overcome the impact of these mismatches on PCR sensitivity, the mismatched nucleotides were replaced with degenerate nucleotides. Oomycete communities from 35 soil samples collected from asymptomatic and root rot diseased sites in pea fields across Alberta were analyzed simultaneously using ITS6–ITS7 and ITS6–ITS7-a.e. (modified version of ITS7) primer sets on 1 Illumina MiSeq run. The number of high-quality reads obtained by ITS6–ITS7-a.e. was more than twice that of ITS6–ITS7. The relative abundance of Pythium spp. was reduced and Aphanomyces spp. increased. Aphanomyces cf. cladogamus and Aphanomyces euteiches were the second and third most abundant species, respectively, in the pea rhizosphere using the ITS7-a.e. primer, but were rare using the ITS7 primer. These results indicate that use of ITS7-a.e. provides a more accurate picture of oomycete communities than ITS7 by enhancing PCR sensitivity to Aphanomyces.

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Ectomycorrhizal fungal communities associated with Larix gemelinii Rupr. in the Great Khingan Mountains, China

PeerJ ◽

10.7717/peerj.11230 ◽

2021 ◽

Vol 9 ◽

pp. e11230

Author(s):

Yonglong Wang ◽

Yanling Zhao ◽

Ying Xu ◽

Jianjun Ma ◽

Busayo Joshua Babalola ◽

...

Keyword(s):

Fungal Community ◽

Geographic Distance ◽

Fungal Communities ◽

Spatial Distance ◽

Its2 Region ◽

Mean Annual Temperature ◽

Ecological Value ◽

Abundant Otus ◽

The Impact ◽

Great Khingan Mountains

Larix gemelinii is an important tree species in the Great Khingan Mountains in Northeast China with a high economic and ecological value for its role in carbon sequestration and as a source of lumber and nuts. However, the ectomycorrhizal (EM) fungal diversity and community composition of this tree remain largely undefined. We examined EM fungal communities associated with L. gemelinii from three sites in the Great Khingan Mountains using Illumina Miseq to sequence the rDNA ITS2 region and evaluated the impact of spatial, soil, and climatic variables on the EM fungal community. A total of 122 EM fungal operational taxonomic units (OTUs) were identified from 21 pooled-root samples, and the dominant EM fungal lineages were /tricholoma, /tomentella-thelephora, /suillus-rhizopogon, and /piloderma. A high proportion of unique EM fungal OTUs were present; some abundant OTUs largely restricted to specific sites. EM fungal richness and community assembly were significantly correlated with spatial distance and climatic and soil variables, with mean annual temperature being the most important predictor for fungal richness and geographic distance as the largest determinant for community turnover. Our findings indicate that L. gemelinii has a rich and distinctive EM fungal community contributing to our understanding of the montane EM fungal community structure from the perspective of a single host plant that has not been previously reported.

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A Theoretical Analysis of Relations between Pressure Changes along Xylem Vessels and Propagation of Variation Potential in Higher Plants

Plants ◽

10.3390/plants10020372 ◽

2021 ◽

Vol 10 (2) ◽

pp. 372

Author(s):

Ekaterina Sukhova ◽

Elena Akinchits ◽

Sergey V. Gudkov ◽

Roman Y. Pishchalnikov ◽

Vladimir Vodeneev ◽

...

Keyword(s):

Higher Plants ◽

Initial Point ◽

Electrical Signal ◽

Terminal Point ◽

Hydraulic Pressure ◽

Long Distance ◽

Variation Potential ◽

Damaged Zone ◽

Pressure Dynamics ◽

The Impact

Variation potential (VP) is an important long-distance electrical signal in higher plants that is induced by local damages, influences numerous physiological processes, and participates in plant adaptation to stressors. The transmission of increased hydraulic pressure through xylem vessels is the probable mechanism of VP propagation in plants; however, the rates of the pressure transmission and VP propagation can strongly vary. We analyzed this problem on the basis of a simple mathematical model of the pressure distribution along a xylem vessel, which was approximated by a tube with a pressure gradient. It is assumed that the VP is initiated if the integral over pressure is more than a threshold one, taking into account that the pressure is transiently increased in the initial point of the tube and is kept constant in the terminal point. It was shown that this simple model can well describe the parameters of VP propagation in higher plants, including the increase in time before VP initiation and the decrease in the rate of VP propagation with an increase in the distance from the zone of damage. Considering three types of the pressure dynamics, our model predicts that the velocity of VP propagation can be stimulated by an increase in the length of a plant shoot and also depends on pressure dynamics in the damaged zone. Our results theoretically support the hypothesis about the impact of pressure variations in xylem vessels on VP propagation.

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Are Wildfires a Threat to Fungi in European Pinus Forests? A Case Study of Boreal and Mediterranean Forests

Forests ◽

10.3390/f10040309 ◽

2019 ◽

Vol 10 (4) ◽

pp. 309 ◽

Cited By ~ 3

Author(s):

Iván Franco-Manchón ◽

Kauko Salo ◽

Juan Oria-de-Rueda ◽

José Bonet ◽

Pablo Martín-Pinto

Keyword(s):

Boreal Forests ◽

Fungal Diversity ◽

Community Dynamics ◽

Forest Products ◽

Climatic Conditions ◽

Fungal Communities ◽

Mediterranean Forests ◽

Natural Forests ◽

Edible Fungi ◽

Pinus Species

Natural forests and plantations of Pinus are ecologically and economically important worldwide, producing an array of goods and services, including the provision of non-wood forest products. Pinus species play an important role in Mediterranean and boreal forests. Although Pinus species seem to show an ecological adaptation to recurrent wildfires, a new era of mega fires is predicted, owing to climate changes associated with global warming. As a consequence, fungal communities, which are key players in forest ecosystems, could be strongly affected by these wildfires. The aim of this study was to observe the fungal community dynamics, and particularly the edible fungi, in maritime (Pinus pinaster Ait.), austrian pine (Pinus nigra J.F. Arnold), and scots pine (Pinus sylvestris L.) forests growing under wet Mediterranean, dry Mediterranean, and boreal climatic conditions, respectively, by comparing the mushrooms produced in severely burned Pinus forests in each area. Sporocarps were collected during the main sampling campaigns in non-burned plots, and in burned plots one year and five years after fire. A total of 182 taxa, belonging to 81 genera, were collected from the sampled plots, indicating a high level of fungal diversity in these pine forests, independent of the climatic conditions. The composition of the fungal communities was strongly affected by wildfire. Mycorrhizal taxa were impacted more severely by wildfire than the saprotrophic taxa, particularly in boreal forests—no mycorrhizal taxa were observed in the year following fire in boreal forests. Based on our observations, it seems that fungal communities of boreal P. sylvestris forests are not as adapted to high-intensity fires as the Mediterranean fungal communities of P. nigra and P. pinaster forests. This will have an impact on reducing fungal diversity and potential incomes in rural economically depressed areas that depend on income from foraged edible fungi, one of the most important non-wood forest products.

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