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Colletotrichum lini is a pathogenic fungus that infects flax and causes significant yield losses. In this study, we assembled the genomes of four highly virulent C. lini strains using the Oxford Nanopore Technologies (ONT, R10.4.1 flow cells) and Illumina platforms. The performance of two tools developed for telomere-to-telomere (T2T) genome assembly was compared: Verkko and Hifiasm. Prior to the assembly, ONT reads were corrected using the HERRO algorithm. Verkko generated genome assemblies of high completeness but low contiguity, while Hifiasm allowed the generation of T2T assemblies. Despite significantly different genome coverage with ONT data (25-100×), four assemblies of equal contiguity were obtained: 53.6-54.7 Mb, ten core chromosomes, and two or three accessory chromosomes. A comparative analysis of different polishing tools showed that at a certain genome coverage with the corrected ONT data (≥35×), the additional polishing of the assembly did not improve its accuracy, even with the Illumina data. An analysis of the genome structures of the four C. lini strains revealed a high similarity between the core chromosomes. Thus, our approach enabled assembling T2T Colletotrichum genomes only from the ONT data obtained using R10.4.1 flow cells and may be promising for other fungal genera. These assemblies will allow the accurate identification of strain-specific differences at the chromosome level and will aid in the development of effective strategies to protect flax from anthracnose.

Colletotrichum lini is a fungal pathogen of flax that can cause significant yield and quality losses. In this work, we obtained the first complete annotated genome assembly of the highly virulent C. lini strain #394-2. The nuclear genome consisted of ten core and two accessory chromosomes and had a length of 53.7 Mb. The mitochondrial genome was 39.1 kb. The assembly was obtained by the Canu-Racon ×2-Medaka-Polca algorithm using Oxford Nanopore Technologies and Illumina data. As a result of the annotation with the Illumina RNA-Seq data, 12,449 genes were identified. Potential signaling proteins were tested for effector functions and 550 effector proteins were predicted using EffectorP. The visualization of the effector protein localization revealed that the presence of effector proteins was associated with repeat-rich regions. A comparison of the genomic structure of C. lini with chromosome-level and complete assemblies of the genus Colletotrichum representatives revealed that the genomes of Colletotrichum species differed by the presence of chromosomal rearrangements. The obtained assembly expands the knowledge of the genomic structure of Colletotrichum species and provides the basis for further studies of C. lini , which will help to understand the virulence mechanisms and protect flax from anthracnose.

Colletotrichum lini is a flax fungal pathogen. The genus comprises differently virulent strains, leading to significant yield losses. However, there were no attempts to investigate the molecular mechanisms of C. lini pathogenicity from high-quality genome assemblies until this study. In this work, we sequenced the genomes of three C. lini strains of high (#390-1), medium (#757), and low (#771) virulence. We obtained more than 100× genome coverage with Oxford Nanopore Technologies reads (N50 = 12.1, 6.1, 5.0 kb) and more than 50× genome coverage with Illumina data (150 + 150 bp). Several assembly strategies were tested. The final assemblies were obtained using the Canu-Racon ×2-Medaka-Polca scheme. The assembled genomes had a size of 54.0-55.3 Mb, 26-32 contigs, N50 values > 5 Mb, and BUSCO completeness > 96%. A comparative genomic analysis showed high similarity among mitochondrial and nuclear genomes. However, a rearrangement event and the loss of a 0.7 Mb contig were revealed. After genome annotation with Funannotate, secreting proteins were selected using SignalP, and candidate effectors were predicted among them using EffectorP. The analysis of the InterPro annotations of predicted effectors revealed unique protein categories in each strain. The assembled genomes and the conducted comparative analysis extend the knowledge of the genetic diversity of C. lini and form the basis for establishing the molecular mechanisms of its pathogenicity.

Flax ( Linum usitatissimum L.) is attacked by numerous devastating fungal pathogens, including Colletotrichum lini , Aureobasidium pullulans , and Fusarium verticillioides ( Fusarium moniliforme ). The effective control of flax diseases follows the paradigm of extensive molecular research on pathogenicity. However, such studies require quality genome sequences of the studied organisms. This article reports on the approaches to assembling a high-quality fungal genome from the Oxford Nanopore Technologies data. We sequenced the genomes of C. lini , A. pullulans , and F. verticillioides ( F. moniliforme ) and received different volumes of sequencing data: 1.7 Gb, 3.9 Gb, and 11.1 Gb, respectively. To obtain the optimal genome sequences, we studied the effect of input data quality and genome coverage on assembly statistics and tested the performance of different assembling and polishing software. For C. lini , the most contiguous and complete assembly was obtained by the Flye assembler and the Homopolish polisher. The genome coverage had more effect than data quality on assembly statistics, likely due to the relatively low amount of sequencing data obtained for C. lini . The final assembly was 53.4 Mb long and 96.4% complete (according to the glomerellales_odb10 BUSCO dataset), consisted of 42 contigs, and had an N50 of 4.4 Mb. For A. pullulans and F. verticillioides ( F. moniliforme ), the best assemblies were produced by Canu-Medaka and Canu-Homopolish, respectively. The final assembly of A. pullulans had a length of 29.5 Mb, 99.4% completeness (dothideomycetes_odb10), an N50 of 2.4 Mb and consisted of 32 contigs. F. verticillioides ( F. moniliforme ) assembly was 44.1 Mb long, 97.8% complete (hypocreales_odb10), consisted of 54 contigs, and had an N50 of 4.4 Mb. The obtained results can serve as a guideline for assembling a de novo genome of a fungus. In addition, our data can be used in genomic studies of fungal pathogens or plant-pathogen interactions and assist in the management of flax diseases.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

In the present work, a highly pathogenic isolate of Fusarium oxysporum f. sp. lini , which is the most harmful pathogen affecting flax ( Linum usitatissimum L.), was sequenced for the first time. To achieve a high-quality genome assembly, we used the combination of two sequencing platforms - Oxford Nanopore Technologies (MinION system) with long noisy reads and Illumina (HiSeq 2500 instrument) with short accurate reads. Given the quality of DNA is crucial for Nanopore sequencing, we developed the protocol for extraction of pure high-molecular-weight DNA from fungi. Sequencing of DNA extracted using this protocol allowed us to obtain about 85x genome coverage with long (N50 = 29 kb) MinION reads and 30x coverage with 2 × 250 bp HiSeq reads. Several tools were developed for genome assembly; however, they provide different results depending on genome complexity, sequencing data volume, read length and quality. We benchmarked the most requested assemblers (Canu, Flye, Shasta, wtdbg2, and MaSuRCA), Nanopore polishers (Medaka and Racon), and Illumina polishers (Pilon and POLCA) on our sequencing data. The assembly performed with Canu and polished with Medaka and POLCA was considered the most full and accurate. After further elimination of redundant contigs using Purge Haplotigs, we achieved a high-quality genome of F. oxysporum f. sp. lini with a total length of 59 Mb, N50 of 3.3 Mb, and 99.5% completeness according to BUSCO. We also obtained a complete circular mitochondrial genome with a length of 38.7 kb. The achieved assembly expands studies on F. oxysporum and plant-pathogen interaction in flax., (Copyright © 2020 Krasnov, Pushkova, Novakovskiy, Kudryavtseva, Rozhmina, Dvorianinova, Povkhova, Kudryavtseva, Dmitriev and Melnikova.)

Being a valuable agricultural plant, flax ( Linum usitatissimum L.) is used for oil and fiber production. However, the cultivation of this agriculture faces an urgent problem of flax susceptibility to fungal diseases. The most destructive ones are caused by the representatives of Fusarium, Colletotrichum, Aureobasidium, Septoria , and Melampsora genera, reducing flax yields significantly. To combat such pathogens effectively, it is of high importance to assess their genetic diversity that can be used to develop molecular markers to distinguish fungal genera and species. Morphological analysis traditionally carried out for fungal identification requires a given amount of time and tends to be difficult. In the present work, we determined the DNA sequences that are frequently used for phylogenetic studies in fungi - internal transcribed spacer (ITS) and beta-tubulin ( tub2 ), translation elongation factor 1-alpha ( tef1 ), RNA polymerase II largest subunit ( RPB1 ), RNA polymerase II second largest subunit ( RPB2 ), and minichromosome maintenance protein ( MCM7 ) genes - for 203 flax fungal pathogens of Fusarium oxysporum, F. avenaceum, F. solani, F. sporotrichiella, F. moniliforme, F. culmorum, F. semitectum, F. gibbosum, Colletotrichum lini, Aureobasidium pullulans, Septoria linicola , and Melampsora lini species. The sequencing was performed using the Illumina MiSeq platform with a 300+300 bp kit, and on average, about 2350 reads per sample were obtained that allows accurate identification of the genetic polymorphism. Raw data are stored at the Sequence Read Archive under the accession number PRJNA596387. The obtained data can be used for fungal phylogenetic studies and the development of a PCR-based test system for flax pathogen identification., (© 2020 The Author(s).)

The present study analysed the response of whitefish (Coregonus lavaretus L.) to reduced toxicity after the long-term contamination of subarctic Lake Imandra. High concentrations of Ni, Al, and Sr in fish organs and tissues were accompanied by nephrocalcinosis, scoliosis, and myopathy during the period of intense contamination. After reduction of the toxic impact on the lake, the accumulation of Cu, Al, Sr, Cr, Pb, and Hg in the kidney, which is the target organ for toxicity, was two-fold less and that of Cd was 10-fold less in whitefish from the contaminated part of the lake compared with those in whitefish from the non-contaminated parts of the lake. The ecological success of whitefish from the contaminated part of the lake was associated with the limited accumulation of metals in organs and tissues and a more favourable physiological state compared with whitefish from non-contaminated parts of the lake. Redistribution models were constructed for essential and non-essential metals in the fish liver and kidney depending on the physiological state of fish. The results revealed changes in Fe and Zn metabolism: an increase in Fe and a decrease in Zn accumulation in the liver with increasing stage of liver disease and decreasing blood haemoglobin concentration. Furthermore, under the sub-toxic (except for Cu) conditions of Lake Imandra, the strategy of adaptive fish response is to preserve and maintain acid-base regulation system., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A comprehensive assessment is presented of the ecotoxicological situation in the Volga River basin from the viewpoint of ecosystem health. Concentrations of organic and inorganic toxic substances in water are reported. Basic clinical and postmortem signs of fish intoxication are described; changes in the cellular structure of their organs and tissues, as well as disturbances in hemogenesis, developing under the effect of toxic agents, are characterized. The comparative characteristics of several microelements (Ni, Cu, Sr, Al, Zn, Co, Mn, Pb, Cd, Hg, As) found in fish are presented. The main disturbances to fish caused by the accumulation of microelements in their organs and tissues are also considered. Based on dose-effect dependencies calculated with respect to the total concentration of toxic substances, standardized to MPC, and fish health criteria, cases that exceed the critical levels of pollutants are demonstrated for the investigated river sections.

Throughout the Kola region of Russia there has been a substantial increase of metal concentrations in water, which are related to local discharges from metallurgical and mining industry, transboundary transmissions as well as indirect leaching of elements by acid precipitation. This study presents data on the levels of Ni, Cu, Sr, Al, Zn, Co, Mn, Pb, Cd, Hg in the organs and tissues of fish, and evaluates relationships with water chemistry. Special attention is paid to fish pathologies, whose aetiology is related to the accumulation of metals and the associated changes of the elementary ratios within the organism. Ecotoxicological assessment of the copper nickel, strontium and acidification regimes also is considered in this article. In general we observed a large number of lakes that are heavily contaminated by Ni and Cu. Fish in these lakes contain high concentrations of Ni and Cu and display frequent pathologies, mostly associated with the kidneys. In lakes contaminated with Sr, there also are high Sr levels in fish and pathologies associated with skeletal tissues. Exposure to acidified water appears to increase the transport of metals (including Al, Ni and Cu) into fish and hence the toxic effects.