April 21, 2020  |  

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Whole Genome Sequencing and Analysis of Chlorimuron-Ethyl Degrading Bacteria Klebsiella pneumoniae 2N3.

Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ?ORF 0934, ?ORF 0492, and wild type (WT) reached their highest growth rates after 8-10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ?ORF 0934 and ?ORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ?ORF 0934, ?ORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.


April 21, 2020  |  

Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay.

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other’s closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit-fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.


April 21, 2020  |  

Intercellular communication is required for trap formation in the nematode-trapping fungus Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a large and diverse group of fungi, which may switch from a saprotrophic to a predatory lifestyle if nematodes are present. Different fungi have developed different trapping devices, ranging from adhesive cells to constricting rings. After trapping, fungal hyphae penetrate the worm, secrete lytic enzymes and form a hyphal network inside the body. We sequenced the genome of Duddingtonia flagrans, a biotechnologically important NTF used to control nematode populations in fields. The 36.64 Mb genome encodes 9,927 putative proteins, among which are more than 638 predicted secreted proteins. Most secreted proteins are lytic enzymes, but more than 200 were classified as small secreted proteins (< 300 amino acids). 117 putative effector proteins were predicted, suggesting interkingdom communication during the colonization. As a first step to analyze the function of such proteins or other phenomena at the molecular level, we developed a transformation system, established the fluorescent proteins GFP and mCherry, adapted an assay to monitor protein secretion, and established gene-deletion protocols using homologous recombination or CRISPR/Cas9. One putative virulence effector protein, PefB, was transcriptionally induced during the interaction. We show that the mature protein is able to be imported into nuclei in Caenorhabditis elegans cells. In addition, we studied trap formation and show that cell-to-cell communication is required for ring closure. The availability of the genome sequence and the establishment of many molecular tools will open new avenues to studying this biotechnologically relevant nematode-trapping fungus.


April 21, 2020  |  

A whole genome scan of SNP data suggests a lack of abundant hard selective sweeps in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum.

The pathogenic fungus Sclerotinia sclerotiorum infects over 600 species of plant. It is present in numerous environments throughout the world and causes significant damage to many agricultural crops. Fragmentation and lack of gene flow between populations may lead to population sub-structure. Within discrete recombining populations, positive selection may lead to a ‘selective sweep’. This is characterised by an increase in frequency of a favourable allele leading to reduction in genotypic diversity in a localised genomic region due to the phenomenon of genetic hitchhiking. We aimed to assess whether isolates of S. sclerotiorum from around the world formed genotypic clusters associated with geographical origin and to determine whether signatures of population-specific positive selection could be detected. To do this, we sequenced the genomes of 25 isolates of S. sclerotiorum collected from four different continents-Australia, Africa (north and south), Europe and North America (Canada and the northen United States) and conducted SNP based analyses of population structure and selective sweeps. Among the 25 isolates, there was evidence for two major population clusters. One of these consisted of 11 isolates from Canada, the USA and France (population 1), and the other consisted of nine isolates from Australia and one from Morocco (population 2). The rest of the isolates were genotypic outliers. We found that there was evidence of outcrossing in these two populations based on linkage disequilibrium decay. However, only a single candidate selective sweep was observed, and it was present in population 2. This sweep was close to a Major Facilitator Superfamily transporter gene, and we speculate that this gene may have a role in nutrient uptake from the host. The low abundance of selective sweeps in the S. sclerotiorum genome contrasts the numerous examples in the genomes of other fungal pathogens. This may be a result of its slow rate of evolution and low effective recombination rate due to self-fertilisation and vegetative reproduction.


April 21, 2020  |  

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes. Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Penicillium purpurogenum Produces a Set of Endoxylanases: Identification, Heterologous Expression, and Characterization of a Fourth Xylanase, XynD, a Novel Enzyme Belonging to Glycoside Hydrolase Family 10.

The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.


April 21, 2020  |  

Assessment of the microbial diversity of Chinese Tianshan tibicos by single molecule, real-time sequencing technology.

Chinese Tianshan tibico grains were collected from the rural area of Tianshan in Xinjiang province, China. Typical tibico grains are known to consist of polysaccharide matrix that embeds a variety of bacteria and yeasts. These grains are widely used in some rural regions to produce a beneficial sugary beverage that is slightly acidic and contains low level of alcohol. This work aimed to characterize the microbiota composition of Chinese Tianshan tibicos using the single molecule, real-time sequencing technology, which is advantageous in generating long reads. Our results revealed that the microbiota mainly comprised of the bacterial species of Lactobacillus hilgardii, Lactococcus raffinolactis, Leuconostoc mesenteroides, Zymomonas mobilis, together with a Guehomyces pullulans-dominating fungal community. The data generated in this work helps identify beneficial microbes in Chinese Tianshan tibico grains.


April 21, 2020  |  

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34?Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.


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