Internal Transcribed Spacer Barcode Research Articles

A Specific Resistance Gene to Leptosphaeria maculans, Rlm11, Has a Limited Impact on Brassica napus Mycobiota Compared with Plant Compartment or Cropping Season Effects

One important ecological question regarding the use of plant resistance genes against fungal pathogens concerns whether and how such resistance genes may modify pathogenic or beneficial members of the plant-associated microbiota. We studied the impact of a plant resistance gene by analyzing the mycobiota associated with Brassica napus organs over two cropping seasons. Sampling dates coincided with key stages of the life cycle of the B. napus pathogen Leptosphaeria maculans. Leaf samples were collected at three time points in autumn and spring, and stem base samples were collected at two time points a few weeks before and at harvest. Stem residues, where L. maculans survives in the intercropping season and develops sexual reproduction, were also analyzed at four time points between the two cropping seasons. The sampling was performed on two plant genotypes, Darmor and Darmor- Rlm11, only differing by the effective resistance gene against L. maculans, Rlm11. Altogether, 419 samples were analyzed using two barcode: internal transcribed spacer (ITS) and Actin. The plant organ was shown to be the main mycobiota structuring factor, as clear-cut alternation of the species suggested that each plant organ represented a specific ecological niche. The cropping season and plant genotype also significantly influenced the community structure in lower proportions. The resistance gene contributed differently to the community structure depending on the year and the organ concerned. A significant but low impact of Rlm11 on other B. napus fungal pathogens was detected. The ITS and Actin barcodes showed similar results, but the species assignation was limited for the latter.

First report of Bot Canker (Diplodia corticola) in Pin Oak (Quercus palustris) in Tennessee.

Diplodia corticola is a fungal pathogen contributing to oak (Quercus spp.) decline in the Mediterranean and US (Félix et al., 2017; Ferreira et al., 2021). In 2021, this pathogen was detected in Tennessee (TN) causing branch dieback in Q. alba (Onufrak et al., 2022). In September 2021, a matured pin oak (Q. palustris) with wilted leaves and elongated branch cankers was observed in the State Botanical Garden of Tennessee-Knoxville (TN, US). Small sections of the phloem were sampled from canker margins of a symptomatic branch using a sterile scalpel, surface sterilized, and plated onto potato dextrose agar amended with antibiotics (PDA++) (Gazis et al. 2018). Three days later, a fungal isolate resembling D. corticola was cultured on ½ PDA. Diplodia corticola is characterized on half-strength PDA by fast growth, irregular margins, and dense white mycelium that turns dark, grayish as the mycelium matures (Úrbez-Torres et al., 2010; Alves et al., 2004). Total genomic DNA was extracted from this isolate following Gazis et al. (2018), and the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and transcription elongation factor 1-α (ef1-α) were amplified (Ferreira et al. 2021). Resulting PCR products were sequenced and assembled into consensus sequences using Unipro UGENE v. 44.0 (Okonechnikov et al., 2012). Each consensus sequence identity was determined using BLAST on the NCBI nucleotide database, restricted to type material. The ITS (accession OQ189888), ef-1α (accession OQ201608), and LSU (accession OQ189887) sequences had a 99.6% (accession KF766156.1), 98.6% (accession XM_020275852.1), and 100% (accession KF766323.1) identity match with D. corticola type culture CBS112549, respectively. To complete Koch's postulates and assess potential pathogenicity on economically and ecologically relevant oaks, 10 pin (Q. palustris; caliper 15.6 ± 2.0 mm), 10 overcup (Q. lyrata; caliper 15.1 ± 2.4 mm), and 10 sawtooth (Q. acutissima; 16.1 ± 2.1 mm) oaks were acclimated in the greenhouse for 1 week prior to the experiment. Five trees of each species were then randomly inoculated at 30 cm above the soil line with a 3 mm diameter plug of D. corticola (grown for 10 days on PDA; Sitz et al. 2017). To serve as a control, the remaining 5 trees for each species received a 3 mm diameter PDA plug. Fifteen days post-inoculation, seepage was observed in D. corticola-inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks. No seepage from wound sites was noted in control trees. Cankers were exposed, photographed, and then measured using ImageJ (Rasband, 2012). Using a sterile scalpel, four wood chips were excised from canker margins and plated onto PDA++. We recovered D. corticola from symptomatic inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks and confirmed species identity by extracting DNA and amplifying the ITS, ef-1α, and LSU regions as described above (Gazis et al., 2018; Ferreira et al., 2021). The resulting consensus sequences matched the D. corticola type culture (CBS112549) ITS (99.0%-99.8% identity), ef-1α (91.0%-99.1% identity), and LSU (96.9%-100% identity) barcoding regions. Cankers were significantly larger in D. corticola-inoculated pin (4.7 ± 1.5 cm2; P = 0.003), overcup (6.8 ± 2.9 cm2; P = 0.009), and sawtooth (5.1 ± 1.3 cm2; P = 0.001) oaks in comparison to the control trees from these groups. Based on current reports, this is the first record of D. corticola causing dieback in pin oak (Q. palustris) in TN.

An accident waiting to happen? Exposing the potential of urogenital schistosomiasis transmission in the Lake Albert region, Uganda

BackgroundUrogenital schistosomiasis caused by the parasitic blood fluke Schistosoma haematobium is the most common form of that constitutes a majority of over 240 million schistosomiasis cases. The enigmatic absence of urogenital schistosomiasis in Uganda has, until now, been attributed to the absence of substantial populations of suitable snail intermediate hosts.MethodsMalacological surveys were carried out in 73 sites southeast of Lake Albert, Uganda in October and November 2020. Collected snails were transported to the laboratory for identification. The snails were identified using partial mitochondrial cytochrome c oxidase subunit one and nuclear internal transcribed spacer barcoding. Schistosome infections in snails were also assessed using cercarial shedding and rapid diagnostic PCR techniques.ResultsWe found Bulinus globosus and Bulinus nasutus productus, the main intermediate species in the transmission of S. haematobium in mainland East Africa. In this survey, B. globosus was more common than B. nasutus productus, with the former reported at four sites (total count = 188) and the latter reported at one site (total count = 79). Molecular testing revealed a high prevalence of Schistosoma bovis in B. nasutus productus (16%), but no S. haematobium infections were found.ConclusionsGiven the abundance of snail hosts and the risky human water contact behaviours observed, we highlight the potential for urogenital schistosomiasis transmission in the region.Graphical

Chromosome-level genome assemblies of Cutaneotrichosporon spp. (Trichosporonales, Basidiomycota) reveal imbalanced evolution between nucleotide sequences and chromosome synteny

BackgroundSince DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity.ResultsThe long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces.ConclusionsOur results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

Applying an internal transcribed spacer as a single molecular marker to differentiate between Tetraselmis and Chlorella species.

In the realm of applied phycology, algal physiology, and biochemistry publications, the absence of proper identification and documentation of microalgae is a common concern. This poses a significant challenge for non-specialists who struggle to identify numerous eukaryotic microalgae. However, a promising solution lies in employing an appropriate DNA barcoding technique and establishing comprehensive databases of reference sequences. To address this issue, we conducted a study focusing on the molecular characterization and strain identification of Tetraselmis and Chlorella species, utilizing the internal transcribed spacer (ITS) barcode approach. By analyzing the full nuclear ITS region through the Sanger sequencing approach, we obtained ITS barcodes that were subsequently compared with other ITS sequences of various Tetraselmis and Chlorella species. To ensure the reliability of our identification procedure, we conducted a meticulous comparison of the DNA alignment, constructed a phylogenetic tree, and determined the percentage of identical nucleotides. The findings of our study reveal the significant value of the ITS genomic region as a tool for distinguishing and identifying morphologically similar chlorophyta. Moreover, our results demonstrate that both the ITS1 and ITS2 regions are capable of effectively discriminating isolates from one another; however, ITS2 is preferred due to its greater intraspecific variation. These results underscore the indispensability of employing ITS barcoding in microalgae identification, highlighting the limitations of relying solely on morphological characterization.

Identification and characterization of Pythium, Globisporangium, and Phytopythium species present in floricultural crops from Long Island, New York.

Several Pythium, Globisporangium and Phytopythium species cause Pythium diseases in greenhouse floricultural crops, resulting in significant seasonal losses. Four hundred eighteen Pythium, Globisporangium and Phytopythium isolates from flowering crops, growing media, or bench and floor debris were collected from Long Island greenhouses or clinic samples between 2002 and 2013. Isolates were identified to species based on morphology and ITS barcoding. Twenty-two species of Pythium, Phytopythium and Globisporangium were identified, with Globisporangium irregulare sensu lato being the most common. To determine the origin of inoculum during the 2011 cropping season, eleven microsatellite loci were analyzed in 124 G. irregulare s.l. isolates collected in four greenhouses and six previously collected from clinic samples. Cluster analyses grouped G. irregulare s.l. isolates into four groups: G. irregulare sensu stricto, plus three G. cryptoirregulare clusters. Population structure defined by greenhouse and host was found in two clades. Additionally, the population dynamics of G. irregulare s.l. isolates associated with Pelargonium spp. from 2011 to 2013 were examined using 85 isolates and nine informative microsatellite loci, to assess inoculum survival over multiple cropping seasons. Although most isolates had unique genotypes, closely related genotypes were found in the same locations over different years. Our results indicate that G. irregulare s.l. inocula have local as well as remote origins. Isolates may be initially brought into ornamental operations from common sources, such as infected plant materials or infested potting mixes. Our results support the hypothesis that established strains can serve as inocula and survive in greenhouse facilities over multiple seasons.

Potential application of DNA barcoding technology for identification of medicinal plant species traded in Nairobi County, Kenya

There exists a long history of herbal products use but recently there are concerns on authenticity and safety of some these products. Majority of the herbal products traded in Kenyan markets are processed plant parts, which have been dried or in powder form and are extremely difficult to identify taxonomically. This study was carried out in selected markets in Nairobi County, the capital city of Kenya to assess authenticity of herbal products traded in the region using deoxyribonucleic acid (DNA) barcoding technology by applying internal transcribed spacer (ITS) and ribulose -1,5-bisphosphate carboxylase Large subunit (rbcL) markers. One hundred and twelve herbal products were purchased from practicing herbalists and analyzed using ITS and rbcL DNA barcoding markers. DNA was extracted from acquired samples, amplified and sequences generated. BLAST search with edited generated sequences against the NCBI database was done to recover the expected species. Our findings showed that 35% of the herbal products were wrongly labeled and contained different species compared to those listed on the labels. Out of 92 samples with positive sequences, 58 matched their expected species after the BLAST search. Among the authentic species identified are O. lanceolata, M. oleifera, W. ugandensis, O. europaea, Acacia species, and C. spinarum. However, 32 species did not match the expected species as listed on labels and were concluded to have been substituted. Among them were R. prinoides, T. abyssinica, U. massaica and A. indica, which were substituted with their close relatives, while R. tridentate, Z. usambarense and Prunus africana were substituted with unrelated species with no reported medical benefits. ITS marker had remarkable identification efficiency at 98% to species level compared to rbcL at 89%. We recommend further studies to establish at what stage substitution takes place, as well as utilization of DNA barcoding technology for medicinal plants authentication and verification

DNA-Barcoding Identification of Plant Pathogens for Disease Diagnostics.

The accurate identification of plant pathogens is a critical step to prevent their spread and attenuate their impact. Among the wide range of methods available, DNA-barcoding, i.e., the identification of an organism through the PCR amplification and sequencing of a single locus, remains one of the most straightforward and accurate plant-pathogen identification techniques that can be used in a generic molecular biology lab. This chapter provides a detailed protocol for the isolation of genomic DNA of fungal and oomycete pathogens from fresh field samples and the amplification and sequencing of the internal transcribed spacer (ITS) locus for DNA-barcoding purpose. Amendments to the protocol are provided to help in resolving issues related to the analysis of complicated samples and to the lack of species resolution that can be encountered with ITS barcodes.

Characterization, potential and conservation of Pisang Kates (Musa cv. ABB), a unique local banana cultivar from Pasuruan, East Java, Indonesia

Abstract. Hapsari L, Damaiyani J, Yulistyarini T, Auliya I, Gusmiati LH, Zaro RM. 2022. Characterization, potential and conservation of Pisang Kates (Musa cv. ABB), a unique local banana cultivar from Pasuruan, East Java, Indonesia. Biodiversitas 23: 3521-3532. Characterization of local banana (Musa spp.) germplasm is fundamental for identification and provides information on traits that support their sustainable conservation and optimum utilization. This study aims to characterize and discuss the potential and conservation of Pisang Kates,a local cultivar originatingfrom Tutur, Pasuruan, East Java, Indonesia. The observed characteristics include morphology, molecular, agronomy and yield performance, leaf anatomy and stomata, pollen morphology and viability, fruit total soluble solids and nutrients. Results showed that Pisang Kates hasa unique fruit shape ovoid to globose, weight of40-150 g, appear solitary and randomly attached directly to peduncle, like papaya. It was molecularly by Internal Transcribed Spacer barcode confirmed as ABB genotype. It is tolerant to drought and banana bunchy top virus, but susceptible to wilts. Leaf anatomy has three palisades and isodiametric polygonal epidermals. Leaf stomata is amphistomatic, tetracytic on adaxial,and paracytic on abaxial. Pollen unit is monad, very large, subspheroidal, monocolpate aperture, granulum ornamentation, and low viability. Fruit moderately sweet with nutrient contents equal to dessert banana. Pisang Kates has potential as one serving consumer size fruit, ornamental fruit plant, female parent for breeding,and pioneer plant for rehabilitation programs. Both ex-situ and on-farm conservation strategies of this valuable local banana genetic resource are encouraged.

Metabarcoding of insect-associated fungal communities: a comparison of internal transcribed spacer (ITS) and large-subunit (LSU) rRNA markers.

Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations and early detection of pathogens and invasive species. Complex communities of fungi are regularly characterised by metabarcoding using the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus, but reliance on a single short sequence fragment limits the confidence of identification. Here we link metabarcoding from the ITS2 and LSU D1-D2 regions to characterise fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type), but identification against the respective reference databases using various taxonomic classifiers revealed poor resolution towards lower taxonomic levels, especially the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, using phylogenetic trees (focused on the epidemiologically important Sordariomycetes) we placed OTUs obtained with either marker relative to reference sequences of the entire rRNA cistron that includes both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. Sensitivity analysis of congruence in both markers suggested the biologically most defensible threshold values for OTU delimitation in Sordariomycetes to be 98% for ITS2 and 99% for LSU D1-D2. Studies of fungal communities using the canonical ITS barcode require corroboration across additional loci. Phylogenetic analysis of OTU sequences aligned to the full rRNA cistron shows higher success rate and greater accuracy of species identification compared to probabilistic taxonomic classifiers.

Morphological characteristics of ectomycorrhizas formed by in vitro synthesis between conifer seedlings and Tuber mycelial strains of the Puberulum clade isolated in Japan.

Seedlings of Pinus densiflora and Abies sachalinensis were inoculated with Tuber mycelial strains of the Puberulum clade in vitro to examine the morphological characteristics of their ectomycorrhizas. Axenically germinated seedlings were inoculated with the mycelia of five taxa from the Puberulum clade and grown in glass jars for 4 mo in an illuminated incubator. The seedlings were successfully colonized by the inoculated Tuber strains, as confirmed by the nuclear ribosomal internal transcribed spacer barcoding of the synthesized ectomycorrhizas. The ectomycorrhizas were characterized by a pale yellow to brown color, short needle-shaped cystidia, and net-like hyphal arrangement, and epidermoid cells on the mantle surface; notably, these features are similar to the ectomycorrhizas of various Puberulum clade members. As the ectomycorrhizas of different Tuber species are indistinguishable by morphological characters, molecular techniques are necessary to identify ectomycorrhizas formed by Tuber species within the Puberulum clade.

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods

Ethnopharmacological relevanceThe roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. Aim of this studyThis study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. Materials and methodsThe sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. ResultsThe plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. ConclusionThe nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

Initial sample processing can influence the soil microbial metabarcoding surveys, revealed by Leucocalocybe mongolica fairy ring ecosystem

In this study, we aimed to investigate the influence of soil preservation approaches, especially cryopreservation and high temperature-drying on the sequencing quality of its microbial community and the background microbial diversity information of fairy ring soil from Leucocalocybe mongolica. Through DNA metabarcoding surveys based on 16S rDNA and ITS (Internal Transcribed Spacer) barcodes, we observed that the bacterial abundance was notably changed when the soil samples were exposed in room temperature for 4 h, whereas the fungal composition was not significantly changed. Moreover, the soil samples preserved their major microbial structures even after high temperature-drying for 12 h, whereas their microbial diversity was influenced. Overall, a total of 9283 and 1871 OTUs (Operational Taxonomic Units) were obtained from soil bacteria and fungi, respectively, and we observed that Chthoniobacteraceae and Tricholomataceae were the dominant bacterial and fungal families in the fairy ring soil, respectively. Our study reveals the impact of soil processing methods on the microbial community compositions contributes to the understanding of fairy ring ecology. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1996272 .

Genetic Differentiation and Divergence Time of Chinese Parnassius (Lepidoptera: Papilionidae) Species Based on Nuclear Internal Transcribed Spacer (ITS) Sequence Data

AbstractParnassius (Lepidoptera: Papilionidae) is a genus of attractive butterflies mainly distributed in the mountainous areas of Central Asia, the Himalayas, and western China. In this study, we used the internal transcribed spacer (ITS1 and ITS2) sequence data as DNA barcodes to characterize the genetic differentiation and conduct the phylogenetic analysis and divergence time estimation of the 17 Parnassius species collected in China. Species identification and genetic differentiation analysis suggest that the ITS barcode is an effective marker for Parnassius species identification; additionally, a relatively high level of genetic diversity and low level of gene flow were detected in the five Parnassius species with diverse geographic populations. Phylogenetic analysis indicates that the 17 species studied were clustered in six clades (subgenera), with subgenus Parnassius at the basal position in the phylogenetic trees. Bayesian divergence time estimation shows that the genus originated about 18 million years ago during the early Miocene, correlated with orogenic events in the distribution region, probably southwestern China about 20–10 million years ago. Our estimated phylochronology also suggests that the Parnassius interspecific and intraspecific divergences were probably related with the rapid rising of the Qinghai-Tibet Plateau, the Tibet Movement, the Kunlun-Yellow River Tectonic Movement, and global cooling associated with intensified glaciation in the region during the Quaternary Period.

Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations

DNA barcoding using the nuclear internal transcribed spacer (ITS) has become prevalent in surveys of fungal diversity. This approach is, however, associated with numerous caveats, including the desire for speed, rather than accuracy, through the use of automated analytical pipelines, and the shortcomings of reference sequence repositories. Here we use the case of a specimen of the bracket fungus Trametes s.lat. (which includes the common and widespread turkey tail, T. versicolor) to illustrate these problems. The material was collected in Vietnam as part of a biodiversity inventory including DNA barcoding approaches for arthropods, plants and fungi. The ITS barcoding sequence of the query taxon was compared against reference sequences in GenBank and the curated fungal ITS database UNITE, using BLASTn and MegaBLAST, and was subsequently analysed in a multiple alignment-based phylogenetic context through a maximum likelihood tree including related sequences. Our results initially indicated issues with BLAST searches, including the use of pairwise local alignments and sorting through Total score and E value, rather than Percentage identity, as major shortcomings of the DNA barcoding approach. However, after thorough analysis of the results, we concluded that the single most important problem of this approach was incorrect sequence labelling, calling for the implementation of third-party annotations or analogous approaches in primary sequence repositories. In addition, this particular example revealed problems of improper fungal nomenclature, which required reinstatement of the genus name Cubamyces (= Leiotrametes), with three new combinations: C. flavidus, C. lactineus and C. menziesii. The latter was revealed as the correct identification of the query taxon, although the name did not appear among the best BLAST hits. While the best BLAST hits did correspond to the target taxon in terms of sequence data, their label names were misleading or unresolved, including [Fungal endophyte], [Uncultured fungus], Basidiomycota, Trametes cf. cubensis, Lenzites elegans and Geotrichum candidum (an unrelated ascomycetous contaminant). Our study demonstrates that accurate identification of fungi through molecular barcoding is currently not a fast-track approach that can be achieved through automated pipelines.Citation: Lucking R., Truong B. V., Huong D. T. T., Le N. H., Nguyen Q. D., Nguyen V. D., Raab-Straube E. von, Bollendorff S., Govers K. & Di Vincenzo V. 2020: Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations. – Willdenowia 50: 383–403. doi: https://doi.org/10.3372/wi.50.50302Version of record first published online on 15 September 2020 ahead of inclusion in December 2020 issue.

DNA barcoding of Pericopsis mooniana from two different populations in Indonesia based on rDNA ITS (Internal Transcribed Spacer)

A DNA barcoding is a taxonomy method that provide fast and acurate species identification, thus it can be applied to anticipate when a species can not be identified morphologically. This study aimed to assess molecular characteristics of Pericopsis mooniana from two wild populations in Indonesia using a barcoding marker rDNA ITS (Internal Transcribed Spacer) as additional information for establishing conservation strategy of this species. Leaf samples of P. mooniana were collected from natural forests at South East Sulawesi and South Kalimantan, and a germplasm orchard at Purwodadi Botanic Garden. Molecular characteristic was evaluated based on a pair of ITS primers that produce between 634-641 bp nucleotide. The sequence data were analyzed by Bioedit version 7.0.5.3 and Mega 7 software. Results showed that P. mooniana from South East Sulawesi and South Kalimantan were clustered together, while P. mooniana from Purwodadi Botanic Garden were separated with only one base difference from those two population. In conclusion, ITS barcoding in this study shows a diffences between P. mooniana collected from South East Sulawesi and South Kalimantan with P. mooniana that originally from Papua. A further study using more barcoding marker and more population could be approached to obtain more information on molecular characters of P. mooniana populations that will useful for establishing conservation and tree breeding program of this species.

Differentiation of Siam weed (Chromolaena odorata (L.) R.M. King & H. Rob.) and morphologically related species in Asteraceae by ITS barcode coupled with high resolution melting analysis (HRM)

Siam weed (Chromolaena odorata (L.) R.M. King & H. Rob.) is a commercially viable plant that has various pharmacological properties including hemostasis and wound healing. Three other plant species as Praxelis (Praxelis clematidea R.M. King & H. Rob.), Goat weed (Ageratum conyzoides L.) and Crofton weed (Ageratina adenophora (Spreng.) R.M. King) in the same family have similar morphological characteristics. Misidentification of processed materials can occur in the absence of a skilled taxonomist. These four plants can often be found in close proximity, leading to erroneous sample collection and misleading results. DNA identification through molecular techniques is an accurate method for correctly identifying and differentiating Siam weed to control the quality of product development. Two primers (ITS1A and ITS1B) were designed based on the barcode (Bar) locus of the internal transcribed spacer (ITS) region. High resolution melting (HRM) analysis (Bar-HRM) was performed to acquire melting shapes and melting temperatures (Tm) to discern the four plant species. Bar-HRM using ITS1A and ITS1B showed differences in melting shapes among the four species. The method employing ITS1A distinguished the four species with higher accuracy than ITS1B. The Tm values of each species obtained from Bar-HRM using ITS1A ranged from 82.54 °C to 86.26 °C. Bar-HRM using ITS served as a rapid and reliable tool for species differentiation among Siam weed, Praxelis, Goat weed and Crofton weed.

Its2vec: Fungal Species Identification Using Sequence Embedding and Random Forest Classification.

Fungi play essential roles in many ecological processes, and taxonomic classification is fundamental for microbial community characterization and vital for the study and preservation of fungal biodiversity. To cope with massive fungal barcode data, tools that can implement extensive volumes of barcode sequences, especially the internal transcribed spacer (ITS) region, are necessary. However, high variation in the ITS region and computational requirements for processing high-dimensional features remain challenging for existing predictors. In this study, we developed Its2vec, a bioinformatics tool for the classification of fungal ITS barcodes to the species level. An ITS database covering more than 25,000 species in a broad range of fungal taxa was assembled. For dimensionality reduction, a word embedding algorithm was used to represent an ITS sequence as a dense low-dimensional vector. A random forest-based classifier was built for species identification. Benchmarking results showed that our model achieved an accuracy comparable to that of several state-of-the-art predictors, and more importantly, it could implement large datasets and greatly reduce dimensionality. We expect the Its2vec model to be helpful for fungal species identification and, thus, for revealing microbial community structures and in deepening our understanding of their functional mechanisms.

Over the hills, but how far away? Estimates of mushroom geographic range extents

AbstractAimGeographic distributions of mushroom species remain poorly understood despite their importance for advancing our understanding of the habitat requirements, species interactions and ecosystem functions of this key group of organisms. Here, we estimate geographic range extents (maximum within‐species geographic distance) of genetically defined operational taxonomic units (OTUs).LocationWorld‐wide, with emphasis on the American Pacific Northwest.TaxaAmanita, Agaricus, Cortinarius, Galerina, Hebeloma, Hydnum, Hygrocybe, Hygrophorus, Inocybe, Lepiota, Pholiota, and Russula + Lactarius; other genera in Agaricomycotina.MethodWe used publicly available OTUs from ribosomal internal transcribed spacer (ITS) sequences (n = 15,373) from 12 mushroom genera with worldwide distributions. For each of 2,324 ~ species‐level OTUs, we estimated the maximum within‐species range extent based on sample locality records. In parallel, we estimated range extents for species in four tree genera. Contrasting estimates from well‐studied trees allowed us to test for potential biases in our range estimates of less well inventoried mushrooms.ResultsThe median range extents across the 2,324 mushroom OTUs varied from ~ 1,200 to 4,039 km, depending on assumptions. These extents were significantly lower than estimates from permuted or randomized data. Mushroom ranges were comparable to the median natural range extent of tree species (1,613 km). In contrast, the tree median species range increased to 16,581 km when anthropogenic range extensions were included. At least 10 mushroom species were similarly broadly distributed, eight of which have been associated with human activity.Main conclusionsOverall, like tree species, mycorrhizal and saprotrophic fungi show evidence of biogeographic structure rather than global distributions. This reconstruction of geographic range extents drew upon investments into ITS barcoding of extensive herbarium collections. Large scale analyses such as ours can yield estimates of fungal geographic range extents that are a prerequisite to a deeper understanding of the diverse roles of fungi in ecosystems.

Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities—A case study

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.