Genomic constitution of diploid species of the genus Avena L. with A-genome according to the data of the next-generation sequencing (NGS)

For diploid (2x) species with the A-genome, as well as for hexaploid (6x) from the genus Avena, a locus-specific next-generation sequencing (NGS) of the sequence of the region of the internal transcribed spacer ITS1 and the beginning of the 5.8S rRNA gene was carried out on the Illumina platform. The high diversity and heterogeneity of the genomes of diploid species are shown. It was revealed that the genomes of modern diploid oat species are relatively far removed from the hexaploid species. It was found that A. canariensis occupies an isolated position among other diploid species, and also takes only an insignificant role in the formation of hexaploid genomes.

Molecular phylogenetic study of rare weed-field species of the genus Avena L.

A molecular phylogenetic study of weed-field species of the genus Avena L. using marker sequences ITS1–5.8S rRNA gene–ITS2 was undertaken. In addition, next-generation sequencing (NGS) was performed on the Illuminaplatform for the ITS1 sequence and the beginning of the gene 5.8S rRNA. Sanger sequencing results revealed the separateclade of microspecies with a good level of support and small level of difference between themselves. According to NGSsequencing data, the two most abundant subgenomes in terms of the number of sequences were identified. Among thecommon sequences of hexaploids, those associated with the C-genome were not found. The presence of unique ribotypeswas shown for A. persica and A. georgica.

First report of Didymosphaeria rubi-ulmifolii associated with canker and dieback of apple trees in southern Ethiopia

Cultivation of apple trees in the highlands of Ethiopia began in 1955. In 2014, blistering of the bark due to cankers on the main stems mostly below the grafting points, followed by dieback and eventually death of apple trees, was observed in apple orchards in the Hadiya Zone in Ethiopia. This study aimed to identify the causal agent of canker and dieback symptoms on the apple trees. Symptomatic trunks from 20 trees (ten per cultivar) were sampled. Isolations were performed from ten trunks (five per cultivar). Fungus colonies with similar cultural features were obtained from all the samples, and the morphology of a representative isolate was characterized. Phylogenetic analyses of the concatenated internal transcribed spacers 1 and 2 and 5.8S rRNA gene, large subunit and actin gene regions confirmed the identity of two isolates as Didymosphaeria rubi-ulmifolii. Pathogenicity was confirmed for one isolate by inoculations of healthy branches of ‘Anna’ and ‘Dorsett Golden’ apple trees resulting in lesion formation, and subsequent re-isolation of the inoculated fungus. This study is the first report of D. rubi-ulmifolii associated with dieback of apple trees. This pathogen caused death of more than 26% of apple trees in one commercial orchard, and could cause severe losses for smallholder apple growers in Ethiopia. Future studies are required to assess the magnitude, distribution and management options of this economically important canker disease in Ethiopia.

Comparison of Two Real-Time PCR Assays Targeting Ribosomal Sequences for the Identification of Cystoisospora belli in Human Stool Samples

Cystoisospora (C.) belli is a coccidian parasite associated with acute or chronic gastroenteritis in immunocompromised patients. Dissatisfactory sensitivity of microscopy as the diagnostic standard approach has been described. Here, we comparatively evaluated two real-time PCRs targeting ribosomal RNA gene sequences of C. belli in stool in a test comparison without a reference standard applying latent class analysis. Therefore, 1000 stool samples from Ghanaian HIV (human immunodeficiency virus) patients (n = 905) as well as military returnees from the tropics (n = 95) were assessed by both assays in parallel. After the exclusion of 33 samples showing PCR inhibition, 29 and 33 positive results were recorded with the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively, resulting in an accuracy-adjusted prevalence of 3.2%. Nearly perfect agreement between both assays was indicated by Fleiss’ kappa of 0.933 with sensitivity and specificity of 92.8% and 100% as well as 100% and 99.8% for the 5.8S rRNA gene/ITS-2 sequence PCR and the ITS-2 sequence PCR, respectively. Both assays proved to be suitable for the diagnosis of C. belli in human stool samples with slightly better sensitivity of the ITS-2 sequence assay, while the 5.8S rRNA gene/ITS-2 sequence PCR may be considered for confirmatory testing.

Sequence-based Identification and Classification of Fungi.

This book chapter describes the advantages and limitations of the ITS Region as a universal barcode for fungal identification. The ITS region offers several practical advantages as a universal fungal barcode region. The region encompasses segments that permit resolution at different taxonomic levels as it includes the highly conserved 5.8S rRNA gene, the moderately rapidly evolving ITS2 region and the rapidly evolving ITS1 region, flanked by the highly conserved SSU and LSU genes which permit design of PCR primers that are almost panfungal. Over the last two decades the sequence-based identification of fungi has certainly come of age. The ITS region is universally accepted as the primary fungal barcoding region owing to the high barcode gap with the locus for many groups of fungi. Since the species-resolution power of ITS is poor for certain groups of fungi, and higher-level taxonomic resolution is greater with proteincoding genes, the TEF1α locus has been proposed as the universal secondary barcode region. In addition, the historical problems surrounding the reliability of fungal DNA sequences in centralized repositories are slowly being resolved by the development of an increasing number of publicly accessible, curated databases.

Transgenerational role of seed mycobiome – an endosymbiotic fungal composition as a prerequisite to stress resilience and adaptive phenotypes in Triticum

Illumina-MiSeq next-generation sequencing of ITS 5.8S rRNA gene demonstrated the transgenerational transmission of fungal seed-endophytes (mycobiome) across three consecutive wheat host generations under standard-control and drought conditions in the greenhouse. Drought-stressed plants experienced a positive shift in the seed mycobiome’s composition, moderated by the external acquisition of endophytic Penicillium (E+) at the seed level. Untreated (E−) and unstressed plants harbor a maximal fungal diversity of non-equilibrium ecological communities. While fungal composition in drought-stressed E− plants experienced important fluctuation, E+ plants maintained fungal ecological communities in phase equilibrium across generations. E+ plants hosted a relatively higher abundance of Ascomycota in the 2nd and 3rd seed generations of wheat, whereas higher abundance of Basidiomycota was detected in 1st generation seeds. The dynamic response of ecological communities to environmental stress is conducive to E+ plants’ active recruitment of endosymbiotic consortia in seeds, benefiting host stress resilience and phenotype. In contrast, E− plants showed an erratic distribution of detected OTUs with an increased occurrence of phytopathogens and diminished plant performance under stress. The present study gives insight into the understanding of the seed-mycobiome composition and dynamics with the potential to improve plant host traits in an adverse environment.

Genotyping ofPneumocystis jiroveciiby Use of a New Simplified Nomenclature System Based on the Internal Transcribed Spacer Regions and 5.8S rRNA Gene of the rRNA Operon

ABSTRACTGenotyping based on internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA operon has played an important role in understanding the transmission and epidemiology ofPneumocystis jirovecii, one of the major opportunistic pathogens in individuals with AIDS and other immunocompromised individuals. The widespread use of this typing system has resulted in several problems, including inconsistent genotype nomenclatures, difficult data transferability, and complicated interpretation of the length variation in multiple homopolymeric tracts. The aim of this study was to establish a new, simplified genotype nomenclature system forP. jiroveciibased on the ITS1 and ITS2 sequences. We first analyzed the complete ITS1, 5.8S rRNA gene, and ITS2 sequences (termed ITS1-5.8S-ITS2) in 27 recentP. jiroveciiisolates from China and identified 18 unique genotypes. Subsequently, we performed a comprehensive classification of more than 400 ITS1- and ITS2-related sequences from GenBank and an in-depth evaluation of the length variation of multiple homopolymeric tracts within ITS1-5.8S-ITS2. Integration of the results from these analyses led to a new, simplified genotype nomenclature system including 62 unique ITS1-5.8S-ITS2 genotypes, simply designated types 1 through 62. This new system offers several advantages over traditional ITS1- and ITS2-based typing systems, including a simpler analysis and interpretation process, a higher discriminative power, and no limitation in assigning potential new genotypes. This new system is expected to facilitate the standardization ofP. jiroveciigenotyping and easy data exchanges across different laboratories.

Molecular characterization of Moniezia sichuanensis in captive musk deer (Moschus berezovskii)

The musk deer (Moschus berezovskii) is an economically important species from which musk is extracted and used in perfumes and medicines. Cestodes (parasitic flatworms) of the genus Moniezia are important parasites that infect this endangered species and can cause high mortality in young deer. In 1982, Moniezia (S.) sichuanensis sp. nov. was described from a specimen obtained from wild musk deer. The new species was distinct from the other described species of Moniezia by the sawtooth-shaped interproglottidal glands, the thick vagina and the absence of a cirrus spine. In the present study, 12 cestodes collected from musk deer were examined morphologically and confirmed to be M. sichuanensis. Molecular characterization was performed by amplifying and comparing the internal transcribed spacer 1 (ITS1) and 5.8S rRNA gene (ITS1–5.8S) of ribosomal DNA with available sequences from other Moniezia species. The amplified sequences ranged from 761 to 764 bp and similarity ranged from 98.7–100%, compared to 67.8–92.4% with other Moniezia spp. Construction of a phylogenetic tree using the neighbour-joining method indicated that all 12 ITS1–5.8S sequences formed a single clade, confirming M. sichuanensis as a separate species. This study provides novel molecular insight into M. sichuanensis that could prove useful for future diagnosis and control of monieziasis in musk deer.