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Background The world’s top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA. Results In this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays , with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel. Conclusion The modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.

• It is increasingly evident that the plant microbiome is a strong determinant of plant health. While the ability to manipulate the microbiome in plants and ecosystems recovering from disturbance may be useful, our understanding of the plant microbiome in regenerating plant communities is currently limited. • Using 16S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region amplicon sequencing, we characterized the leaf, stem, fine root, rhizome, and rhizosphere microbiome of < 1-yr-old aspen saplings and the associated bulk soil after a recent high-intensity prescribed fire across a burn severity gradient. • Consistent with previous studies, we found that soil microbiomes are responsive to fire. We extend these findings by showing that certain plant tissue microbiomes also change in response to fire. Differences in soil microbiome compositions could be attributed to soil chemical characteristics, but, generally, plant tissue microbiomes were not related to plant tissue elemental concentrations. Using source tracking modeling, we also show that fire influences the relative dominance of microbial inoculum and the vertical inheritance of the sapling microbiome from the parent tree. • Overall, our results demonstrate how fire impacts plant microbiome assembly, diversity, and composition and highlights potential for further research towards increasing plant fitness and ecosystem recovery after fire events.

Soil fungi play a major role in ecological and biogeochemical processes in forests. Little is known, however, about the structure and richness of different fungal communities and the distribution of functional ecological groups (pathogens, saprobes and symbionts). Here, we assessed the fungal diversity in six different forest soils using tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS-1). No less than 166 350 ITS reads were obtained from all samples. In each forest soil sample (4 g), approximately 30 000 reads were recovered, corresponding to around 1000 molecular operational taxonomic units. Most operational taxonomic units (81%) belonged to the Dikarya subkingdom (Ascomycota and Basidiomycota). Richness, abundance and taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The ITS-1 sequences (73%) analysed corresponded to only 26 taxa. The most abundant operational taxonomic units showed the highest sequence similarity to Ceratobasidium sp., Cryptococcus podzolicus, Lactarius sp. and Scleroderma sp. This study validates the effectiveness of high-throughput 454 sequencing technology for the survey of soil fungal diversity. The large proportion of unidentified sequences, however, calls for curated sequence databases. The use of pyrosequencing on soil samples will accelerate the study of the spatiotemporal dynamics of fungal communities in forest ecosystems.

Incompleteness of reference sequence databases and unresolved taxonomic relationships complicates taxonomic placement of fungal sequences. We developed Protax-fungi, a general tool for taxonomic placement of fungal internal transcribed spacer (ITS) sequences, and implemented it into the PlutoF platform of the UNITE database for molecular identification of fungi. With empirical data on root- and wood-associated fungi, Protax-fungi reliably identified (with at least 90% identification probability) the majority of sequences to the order level but only around one-fifth of them to the species level, reflecting the current limited coverage of the databases. Protax-fungi outperformed the Sintax and Rdb classifiers in terms of increased accuracy and decreased calibration error when applied to data on mock communities representing species groups with poor sequence database coverage. We applied Protax-fungi to examine the internal consistencies of the Index Fungorum and UNITE databases. This revealed inconsistencies in the taxonomy database as well as mislabelling and sequence quality problems in the reference database. The according improvements were implemented in both databases. Protax-fungi provides a robust tool for performing statistically reliable identifications of fungi in spite of the incompleteness of extant reference sequence databases and unresolved taxonomic relationships.

   Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita -induced mushroom poisoning. Key points • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases. Graphical Abstract

Rapid development of high-throughput (HTS) molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA) regions often used in metabarcoding analyses. The internal transcribed spacer (ITS) barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs) at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU) and large subunit (LSU) genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6–38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups.

High biodiversity is regarded as a barrier against biological invasions. We hypothesized that the invasion success of the pathogenic ascomycete Hymenoscyphus fraxineus threatening common ash in Europe relates to differences in dispersal and colonization success between the invader and the diverse native competitors. Ash leaf mycobiome was monitored by high-throughput sequencing of the fungal internal transcribed spacer region (ITS) and quantitative PCR profiling of H. fraxineus DNA. Initiation of ascospore production by H. fraxineus after overwintering was followed by pathogen accumulation in asymptomatic leaves. The induction of necrotic leaf lesions coincided with escalation of H. fraxineus DNA levels and changes in proportion of biotrophs, followed by an increase of ubiquitous endophytes with pathogenic potential. H. fraxineus uses high propagule pressure to establish in leaves as quiescent thalli that switch to pathogenic mode once these thalli reach a certain threshold – the massive feedback from the saprophytic phase enables this fungus to challenge host defenses and the resident competitors in mid-season when their density in host tissues is still low. Despite the general correspondence between the ITS-1 and ITS-2 datasets, marker biases were observed, which suggests that multiple barcodes provide better overall representation of mycobiomes.

Different distance-based threshold selection approaches were used to assess and compare use of the internal transcribed spacer (ITS) region to distinguish among 901 Cortinarius species represented by >3000 collections. Sources of error associated with genetic markers and selection approaches were explored and evaluated using MOTUs from genus and lineage based-alignments. Our study indicates that 1%–2% more species can be distinguished by using the full-length ITS barcode as compared to either the ITS1 or ITS2 regions alone. Optimal threshold values for different picking approaches and genetic marker lengths inferred from a subset of species containing major lineages ranged from 97.0% to 99.5% sequence similarity using clustering optimization and UNITE SH, and from 1% to 2% sequence dissimilarity with CROP. Errors for the optimal cutoff ranged from 0% to 70%, and these can be reduced to a maximum of 22% when excluding species lacking a barcode gap. A threshold value of 99% is suitable for distinguishing species in the majority of lineages in the genus using the entire ITS region but only 90% of the species could be identified using just the ITS1 or ITS2 region. Prior identification of species, lacking barcode gaps and their subsequent separate analyses, maximized the accuracy of threshold approaches. Fungal DNA barcoding. Graphical Abstract Figure. Fungal DNA barcoding.

AFLP markers provide a potential source of phylogenetic information for molecular systematic studies. However, there are properties of restriction fragment data that limit phylogenetic interpretation of AFLPs. These are (a) possible nonindependence of fragments, (b) problems of homology assignment of fragments, (c) asymmetry in the probability of losing and gaining fragments, and (d) problems in distinguishing heterozygote from homozygote bands. In the present study, AFLP data sets of Lactuca s.l. were examined for the presence of phylogenetic signal. An indication of this signal was provided by carrying out tree length distribution skewness (g1) tests, permutation tail probability (PTP) tests, and relative apparent synapomorphy analysis (RASA). A measure of the support for internal branches in the optimal parsimony tree (MPT) was made using bootstrap, jackknife, and decay analysis. Finally, the extent of congruence in MPTs for AFLP and internal transcribed spacer (ITS)-1 data sets for the same taxa was made using the partition homogeneity test (PHT) and the Templeton test. These analytical studies suggested the presence of phylogenetic signal in the AFLP data sets, although some incongruence was found between AFLP and ITS MPTs. An extensive literature survey undertaken indicated that authors report a general congruence of AFLP and ITS tree topologies across a wide range of taxonomic groups, suggesting that the present results and conclusions have a general bearing. In these earlier studies and those for Lactuca s.l., AFLP markers have been found to be informative at somewhat lower taxonomic levels than ITS sequences. Tentative estimates are suggested for the levels of ITS sequence divergence over which AFLP profiles are likely to be phylogenetically informative.

Changes in plants and soils during natural succession have been evaluated, but little is known about the effects of succession on the activities of soil microbes and their interactions with soil erodibility. We conducted a field study on the Chinese Loess Plateau, typical of this semiarid area, to determine the effect of secondary succession on the stability of soil structure against erosion and on the composition of soil fungal communities. Characteristics of plant, soil, and fungal communities were assessed across a 30-yr chronosequence of grassland developed from abandoned cropland. The diversity and composition of the fungal communities were determined using high-throughput sequencing of the internal transcribed spacer. Six grasslands were selected to represent different successional age classes: 0 (cropland), 5, 10, 15, 20, and 30 yr. Short-term decreases (initial 5 yr) in the amounts of soil organic carbon, total nitrogen, available phosphorus, and fungal biomass and in fungal diversity had returned to original levels (i.e., cropland) within 15 yr and were much higher after continued succession. Abandoning cropland for succession caused the soil erodibility (K) decrease and the above-ground coverage, soil nutrient levels, content of larger (>5 mm) water-stable aggregate, mean aggregate weight diameter, and diversity of the fungal communities improvement including arbuscular mycorrhizas (AMF), ectomycorrhizas (EMF), and saprotrophs. The fungal communities were dominated by Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota during the succession. The successional patterns of the plant and fungal communities were similar, although distinct fungal communities were not observed in the two initial stages, suggesting that fungal succession may develop more slowly than plant succession. Plant root biomass, EMF, and soil organic carbon content accounted for most of the variation of soil erodibility (28.6%, 19.5%, and 11.8%, respectively), indicating their importance in shaping soil structure to prevent erosion. Our results demonstrated that abandoning cropland for natural succession could decrease soil erodibility and increase fungal diversity. EMF plays an important role in soil stability against erosion in the Loess Plateau. Abandoning cropland for natural succession should be recommended for alleviating soil erosion and improving the degraded soils in this area.