Internal Transcribed Spacer Primers Research Articles

DNA barcoding for identification of consumer-relevant mushrooms: A partial solution for product certification?

One challenge in the dietary supplement industry is confirmation of species identity for processed raw materials, i.e. those modified by milling, drying, or extraction, which move through a multilevel supply chain before reaching the finished product. This is particularly difficult for samples containing fungal mycelia, where processing removes morphological characteristics, such that they do not present sufficient variation to differentiate species by traditional techniques. To address this issue, we have demonstrated the utility of DNA barcoding to verify the taxonomic identity of fungi found commonly in the food and dietary supplement industry; such data are critical for protecting consumer health, by assuring both safety and quality. By using DNA barcoding of nuclear ribosomal internal transcribed spacer (ITS) of the rRNA gene with fungal specific ITS primers, ITS barcodes were generated for 33 representative fungal samples, all of which could be used by consumers for food and/or dietary supplement purposes. In the majority of cases, we were able to sequence the ITS region from powdered mycelium samples, grocery store mushrooms, and capsules from commercial dietary supplements. After generating ITS barcodes utilizing standard procedures accepted by the Consortium for the Barcode of Life, we tested their utility by performing a BLAST search against authenticate published ITS sequences in GenBank. In some cases, we also downloaded published, homologous sequences of the ITS region of fungi inspected in this study and examined the phylogenetic relationships of barcoded fungal species in light of modern taxonomic and phylogenetic studies. We anticipate that these data will motivate discussions on DNA barcoding based species identification as applied to the verification/certification of mushroom-containing dietary supplements.

Rare or rarely detected? Ceraceosorus guamensis sp. nov.: a second described species of Ceraceosorales and the potential for underdetection of rare lineages with common sampling techniques.

Ceraceosorales is a monotypic order in Ustilaginomycotina. Its namesake, Ceraceosorus bombacis, was described as a phytopathogen of Bombax ceiba in India. In this study, we describe Ceraceosorus guamensis sp. nov., collected on the South Pacific island of Guam, which appears to represent the second isolation of any member of this order in over 40years. Ceraceosorus species are monokaryotic and filamentous in culture, producing conidia on potato dextrose agar. However, both species behave yeast-like when cultured on corn meal agar. The internal transcribed spacer (ITS) region (spanning the ITS1-5.8S-ITS2) in both species of Ceraceosorus is highly heterogeneous containing multiple disparate copies that can vary intragenomically by up to 3.5%. Moreover, this region could not be amplified using the fungal ITS primers most frequently used for culture-independent methods of assessing fungal biodiversity. This fact, combined with the extremely slow growth rates on commonly employed media, may indicate that members of this lineage are potentially underdetected by current sampling methods.

Trypanosoma Infection Rates in Glossina Species in Mtito Andei Division, Makueni County, Kenya.

African Animal Trypanosomiasis (AAT) transmitted cyclically by tsetse fly (Glossina spp.) is a major obstacle to livestock production in the tropical parts of Africa. The objective of this study was to determine the infection rates of trypanosomes in Glossina species in Mtito Andei Division, Makueni County, Kenya. Tsetse fly species, G. longipennis and G. pallidipes, were trapped and DNA was isolated from their dissected internal organs (proboscis, salivary glands, and midguts). The DNA was then subjected to a nested PCR assay using internal transcribed spacer primers and individual trypanosome species were identified following agarose gel electrophoresis. Out of the 117 flies trapped in the area 39 (33.3%) were teneral while 78 (67%) were nonteneral. G. pallidipes constituted the largest percentage of 58% while G. longipennis were 42%. The overall trypanosomes infection rate in all nonteneral Glossina spp. was 11.53% with G. longipennis recording the highest infection rate of 23.08% while G. pallidipes had an infection rate of 5.77%. T. vivax was the most infectious (10.26%) compared to T. congolense (1.28%). Mean apparent densities were strongly positively correlated with infection rates (r = 0.95) confirming the importance of this parameter as an indicator of AAT transmission risk.

DNA barcoding for identification of species in mushrooms: A component of product certification

One challenge in the botanical industry is the confirmation of species identification, and this can be compounded by the processing of the materials before reaching the vendor, such as milling or drying. This can be particularly difficult for samples that contain fungal mycelia, where morphological characteristics do not present sufficient variation to differentiate species. However, monitoring the safety and quality of such products is a requirement for the protection of consumer health. DNA barcoding, a genetic-based species identification system, has become popular in the past decade, and it shows great promise for species level identification of fungi. By using DNA barcoding of nuclear ribosomal internal transcribed spacer (ITS) of the rRNA gene with fungal specific ITS primers, we generated ITS barcodes for two representative mushroom fungi, which are being used by consumers for food and dietary supplement purposes. After generating ITS barcodes utilizing standard procedures accepted by the Consortium for the Barcode of Life, we tested the utility of the ITS by performing a BLAST search against both curated and non-curated public databases. Results demonstrated that the ITS region was able to identify both mushrooms used in the present study to species-level. The BLAST search provided 99% similarity of the query sequence to those available in the public database. We anticipate that this presentation will motivate a discussion on DNA barcoding based species identification, particularly as it applies to the verification/certification of mushrooms containing products.

English

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is the most frequently used marker for distinguishing plant species. Previously published polymerase chain reaction (PCR) primers available for amplifying the ITS region from environmental plant samples provide a high degrees of success while maintaining a broad range of compatibility difficult to discriminate against fungal DNA. Thus, the plant-specific ITS primer design is required to achieve the preferential amplification of plant DNA with discrimination against fungal DNA from environmental plant samples. In this study, two primers, ITS1-F2 and ITS4-R, were newly designed and the specificity of these primers were tested against 14 accessions of Pulsatilla tongkangensis and 2 accessions of Pulsatilla cernua. Our results showed that ITS4-R, when paired with either a universal primer ITS5 or the newly designed primer ITS1-F2, efficiently amplified DNA from environmental P. cernua samples and discriminated against parasitic fungal DNAs, while another newly designed primer ITS1-F2, when paired with either a universal primer ITS4 or ITS4-R, could not preferentially amplify plant DNA or discriminate against fungal DNA. For P. tongkangensis, the dismatch of ITS5 primer resulted in noneffective or wrong amplification when paired with either ITS4-R or ITS4, while ITS1-F2, when paired with ITS4-R and ITS4, efficiently amplified plant DNA and both plant and fungal DNAs, respectively. This result suggested that the combination with ITS1-F2 and ITS4-R were intended to be specific to Pulsatilla species. This study will be particularly useful for detection and analysis of plant sequences from environmental or fungus-associated materials. Key words: Pulsatilla, nrDNA ITS, plant-specific primer design, fungus-associated plant discrimination, molecular identification.

Meeting Report: Fungal ITS Workshop (October 2012)

This report summarizes a meeting held in Boulder, CO USA (19–20 October 2012) on fungal community analyses using ultra-high-throughput sequencing of the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA (rRNA) genes. The meeting was organized as a two-day workshop, with the primary goal of supporting collaboration among researchers for improving fungal ITS sequence resources and developing recommendations for standard ITS primers for the research community.

Quantification of the 16S-23S rRNA internal transcribed spacers of Burkholderia xenovorans strain LB400 using real-time PCR in soil samples

This study establishes a new real-time PCR assay (using SYBR Green™ detection) for the identification and the direct quantification of specific individual Burkholderia xenovorans strain LB400 from DNA samples of soil and sediment. Specific primers were designed to amplify a 190-bp fragment of the 16S-23S rRNA internal transcribed spacers (ITS) from LB400. The specificity of primers was evaluated using 21 strains. The detection limit of the real-time PCR was analysed on soil samples inoculated with LB400 and was of 6 copies (10(5) CFUg(-1) of dry sample). The 16S-23S rRNA ITS primers developed in this work for rapid quantification of LB400 were validated. The assay allowed the quantification of LB400 as pure strain and among the indigenous microbial community in samples of soil and sediment (105-day experiment).

Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

Ultra-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 predicted in silico and 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.

Isolation and molecular identification of β-carotene producing strains of Dunaliella salina and Dunaliella bardawil from salt soil samples by using species- specific primers and internal transcribed spacer (ITS) primers

Dunaliella salina and Dunaliella bardawil are unique species of the genus Dunaliella that produce large amounts of â-carotene when cultivated under appropriate conditions. These include high light intensity, high sodium chloride concentration, nitrate deficiency and extreme temperatures. Under these conditions, only D. salina and D. bardawil can accumulate â-carotene to as much as 10% of the cellular dry weight. Because the morphological characterization is based on the environmental factors, the Dunaliella will change the shape, so identification and differentiation of Dunaliella species by morphology is very difficult. In this research study, we isolated, identified and discriminated the different Dunaliella β-carotene producing strains from salt soil samples, by using 18S rDNA and internal transcribed spacer (ITS) gene sequences. The soil samples were collected from four different provinces of the North Eastern part of Thailand.namely: UdonThani, BuriRam, AmnartCharoen and Chaiyaphum. Among the four isolates, only BuriRam KU01 and UdonThani KU01 were D. salina and D. bardawil, respectively whereas AmnartCharoen KU01 and Chaiyaphum KU01 were not these Dunaliella species. At 4 M NaCl, with deficiency of nitrate (KNO 3 ) and phosphate (KH 2 PO 4 ) in the medium, the D. salina strain BuriRam KU01 produced β-carotene at the level of 56.25 ± 0.97 pg·cell -1 and D. bardawil strain UdonThani KU01 produced β-carotene at the level of 52.91 ± 0.29 pg·cell -1 at the 25th day after inoculation. The 18S rDNA and ITS sequences of D. salina strain BuriRam KU01 and D. bardawil strain UdonThani KU01 were submitted to the National Center for Biotechnology Information (NCBI) database with accession numbers of JN052202, JN052203, JN034031 and JN052204, respectively. By using the species-specific primers and ITS primers the â-carotene producing strains of Dunaliella was identified. Key words : 18S rDNA, β-carotene, carotenoid, Dunaliella bardawil, Dunaliella salina , internal transcribed spacer (ITS), salinity.

High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.

Detection of Laminariaceae Species Based on PCR by Family-specific ITS Primers

To analyze nucleotide sequence encoding internal transcribed spacer (ITS) regions specific to the Laminariaceae family, genomic DNA was isolated from six brown algae species distributed along the east coast of Korea. These included three species from the Laminariaceae family (Agarum clathratum Dumortier, Costaria costata [C. Agardh] Saunders, and Saccharina japonica Areschoug) and two species from the Alariaceae family (Undaria pinnatifida [Harvey] Suringer and Ecklonia cava Kjellman), both in the order Laminariales, and one species from the family Sargassaceae in the order Fucales (Sargassum serratifolium). Based on a sequence analysis of ITS-1 and ITS-2 for A. clathratum, C. costata, and E. cava, oligonucleotides were designed from the regions that showed sequence conservation in Laminariaceae. Following polymerase chain reaction using three sets of primers, amplification of ITS-1 and ITS-2 was detected in reactions using genomic DNA isolated from the species belonging to Laminariaceae, but not from the species belonging to the other families. The results indicate that this method can be used for the detection and identification of Laminariaceae species.

Development of a novel PCR assay based on the 16S–23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR-based methods have been developed for the detection and identification of L.garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S-23S internal transcribed spacer (ITS) region and used for the specific detection of L.garvieae. L.garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false-positive results with non-L.garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L.garvieae in fish and aquaculture environments.

Analytical characterization and structure elucidation of metabolites from Aspergillus ochraceus MP2 fungi

Objective To isolate and characterize the bioactive secondary metabolites from Aspergillus ochraceus ( A. ochraceus) MP2 fungi. Methods The anti bacterial activity of marine sponge derived fungi A. ochraceus MP2 was thoroughly investigated against antagonistic human pathogens. The optimum inhibitory concentration of the fungi in the elite solvent was also determined. The promising extracts that showed good antimicrobial activity were subjected to further analytical separation to get individual distinct metabolites and the eluants were further identified by GC MS instrumental analysis. The molecular characterization of the elite fungal strains were done by isolating their genomic DNA and amplify the internal transcribed spacer (ITS) region of 5.8s rRNA using specific ITS primer. The novelty of the strain was proved by homology search tools and elite sequences was submitted to GENBANK. Results Three bioactive compounds were characterized to reveal their identity, chemical formula and structure. The first elutant was identified as α – Campholene aldehyde with chemical formula C 10 H 16 O and molecular weight 152 Da. The second elutant was identified as Lucenin-2 and chemical formula C 27 H 30 O 16 and molecular weight 610 Da. The third elutant was identified as 6-Ethyloct- 3-yl- 2- ethylhexyl ester with Chemical formula C 26 H 42 O4with molecular weight 418 Da. Conclusions The isolated compounds showed significant antimicrobial activity against potential human pathogens. Microbial secondary metabolites represent a large source of compounds endowed with ingenious structures and potent biological activities.

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.

Isolation and molecular characterization of bioactive secondary metabolites from Callyspongia spp. associated fungi

Objective To isolate and characterize the bioactive secondary metabolite from Callyspongia spp. associated fungi. Methods In vitro antibacterial screening of fungi associated with Callyspongia species, collected from south east coast of India, against selected clinical isolates of bacteria were conducted in this study. The extracts showing good antimicrobial activity were subjected to further analysis to identify the active constituents sponge associated fungi (both biomass and filtrate) with five different solvents. The compound responsible for bioactivity was characterized using Fouvier-transform infrared (FT-IR) and gas chromatography-mass spectrometry (GC-MS) instrumental analysis to identify the functional group and compound. The molecular characterization of the elite fungal strains were done by isolating their genomic DNA and amplify the internal transcribed spacer (ITS) region of 5.8 sr RNA using specific ITS primer. The novelty of the strain was proved by BlastN analysis against non-redundant (NR) database and hence was submitted to GenBank. Results Active compound was Desmethylnomifensine confirmed by GC-MS and the potent fungi was Aspergillus flavus GU815344. Conclusions The isolate exhibits a marked antagonistic activity against potential bacterial pathogens thus illuminating the advanced researches in this decade to focus on clinical pharmacology to identify novel therapeutic targets. The present study depicts a promising scenario to focus on Aspergillus flavus derived compounds which can be easily scaled up for large biomass production and stable formulation as a drug.

광릉요강꽃과 복주머니란의 뿌리에 감염된 난균근균의 특성

광릉요강꽃(Cypripedium japonicum)과 복주머니란(C. macranthum)의 뿌리에서 난 균근균이 확인되었다. 두 종의 난 뿌리에서 균근의 형태적인 특성을 관찰하였다. 분자적인 분석을 통하여 두 종의 뿌리로부터 난균근균을 확인하였다. 뿌리에서 DNA를 추출하여, 담자균류 ITS 지역에 특이적인 프라이머인 ITS1-OF와 ITS4-OF를 사용하여 ITS 지역을 증폭한 후 염기서열을 분석하여 두 식물의 뿌리에 공생하는 난균근균을 동정하였다. 광릉요강꽃의 뿌리에는 3종류의 Tulasnellaceae와 1종의 담자균류가 발견되었고, 복주머니란의 뿌리에서는 2종류의 Tulasnellaceae에 속하는 균이발견되었다. Orchid mycorrhizal fungi(OMF) were examined in roots of the two threatened orchid species, Cypripedium japonicum and C. macranthum. The morphological characteristics of mycorrhizal colonization in the roots of two orchid species were observed. OMF colonized in the roots of two species were identified using molecular analysis. DNA from the root was extracted and amplified internal transcribed spacer(ITS) region using basidiomycete ITS primers, ITS1-OF and ITS4-OF. Four species belonging to Tulasnellaceae in roots of C. japonicum and two species of Tulasnellaceae and one basidiomycetous species was found in roots of C. macranthum.

ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

BackgroundDuring the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates.ResultsSome of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi.ConclusionsWe conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.

Cell cultures from the symbiotic soft coral Sinularia flexibilis

The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cultivation of S. flexibilis cells on different media and cell dissociation methodologies. Mechanical dissociation of coral tissue always yielded the highest number of cells and allowed subsequent cellular growth in all treatments. The best results from chemical dissociation reagents were found with trypsin-ethylene diamine tetraacetic acid. Coral cells obtained from spontaneous dissociation did not grow. Light intensity was found to be important for coral cell culture showing an enduring symbiosis between the cultured cells and their intracellular algae. The Grace's insect medium and Grace's modified insect medium were found to be superior substrates. To confirm the similarity of the cultured cells and those in the coral tissue, a molecular test with Internal Transcribed Spacer primers was performed. Thereby, the presence of similar cells of both the coral cells and zooxanthella in different culture media was confirmed.

Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas

Despite advances owing to molecular approaches, several hurdles still obstruct the identification of fungi forming orchid mycorrhizas. The Tulasnellaceae exhibit accelerated evolution of the nuclear ribosomal operon, causing most standard primers to fail in polymerase chain reaction (PCR) trials. Insufficient sequences are available from well characterized isolates and fruitbodies. Lastly, taxon-specific PCR primers are needed in order to explore the ecology of the fungi outside of the orchid root. Here, progress in overcoming these hurdles is reported. Broad-spectrum basidiomycete internal transcribed spacer (ITS) primers that do not exclude most known Tulasnellaceae are presented. blast searches and empirical PCR tests support their wide utility within the Basidiomycota. Taxon-specific ITS primers are presented targeted to orchid-associated Tulasnella, and a core component of the Thelephora-Tomentella complex. The efficiency and selectivity of these primer sets are again supported by blast searches and empirical tests. Lastly, ITS DNA sequences are presented from several strains of Epulorhiza, Ceratorhiza, Ceratobasidium, Sistotrema, Thanatephorus and Tulasnella that were originally described in the landmark mycorrhizal studies of Currah and Warcup. Detailed phylogenetic analyses reveal some inconsistencies in species concepts in these taxonomically challenging resupinate basidiomycetes, but also help to place several sequences from environmental samples.

Morphological and molecular evidence supporting an arbutoid mycorrhizal relationship in the Costa Ricanpáramo

This study examines evidence for a particular arbutoid mycorrhizal interaction in páramo, a high-altitude neotropical ecosystem important in hydrological regulation but poorly known in terms of its fungal communities. Comarostaphylis arbutoides Lindley (Ericaceae) often forms dense thickets in Central American páramo habitats. Based on phylogenetic classification, it has been suggested that C. arbutoides forms arbutoid mycorrhizae with diverse Basidiomycetes and Ascomycetes; however, this assumption has not previously been confirmed. Based on field data, we hypothesized an arbutoid mycorrhizal association between C. arbutoides and the recently described bolete Leccinum monticola Halling & G.M. Mueller; in this study, we applied a rigorous approach using anatomical and molecular data to examine evidence for such an association. We examined root samples collected beneath L. monticola basidiomes for mycorrhizal structures, and we also compared rDNA internal transcribed spacer (ITS) sequences between mycorrhizal root tips and leaf or basidiome material of the suspected symbionts. Root cross sections showed a thin hyphal sheath and intracellular hyphal coils typical of arbutoid mycorrhizae. DNA sequence comparisons confirmed the identity of C. arbutoides and L. monticola as the mycorrhizal symbionts. In addition, this paper provides additional evidence for the widespread presence of minisatellite-like inserts in the ITS1 spacer in Leccinum species (including a characterization of the insert in L. monticola) and reports the use of an angiosperm-specific ITS primer pair useful for amplifying plant DNA from mycorrhizal roots without co-amplifying fungal DNA.