IMA Genome - F19 : A genome assembly and annotation guide to empower mycologists, including annotated draft genome sequences of Ceratocystis pirilliformis, Diaporthe australafricana, Fusarium ophioides, Paecilomyces lecythidis, and Sporothrix stenoceras.

The pace at which Next Generation Sequence data is being produced continues to accelerate as technology improves. As a result, such data are increasingly becoming accessible to biologists outside of the field of bioinformatics. In contrast, access to training in the methods of genome assembly and annotation are not growing at a similar rate. In this issue, we report on a Genome Assembly Workshop for Mycologists that was held at the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria, South Africa and make available the 12 draft genome sequences emanating from the event. With the aim of making the process of genome assembly and annotation more accessible to biologists, we provide a step-by-step guide to both genome assembly and annotation, intended to encourage and empower mycologists to use genome data in their research.

A high-quality draft genome sequence of Neonectria faginata, causative agent of beech bark disease of Fagus grandifolia.

The draft genome of Neonectria faginata was sequenced with Oxford Nanopore and Illumina 250 bp paired-end sequencing technologies. The assembled genome was 42.9 Mb distributed over 24 contigs, with N50 of 4.4 Mb and 98.6% BUSCO completeness. This genome sequence will aid in understanding N. faginata population structure and ecology.

Population genomic analyses suggest recent dispersal events of the pathogen Cercospora zeina into East and Southern African maize cropping systems.

A serious factor hampering global maize production is gray leaf spot disease. Cercospora zeina is one of the causative pathogens, but population genomics analysis of C. zeina is lacking. We conducted whole-genome Illumina sequencing of a representative set of 30 C. zeina isolates from Kenya and Uganda (East Africa) and Zambia, Zimbabwe, and South Africa (Southern Africa). Selection of the diverse set was based on microsatellite data from a larger collection of the pathogen. Pangenome analysis of the C. zeina isolates was done by (1) de novo assembly of the reads with SPAdes, (2) annotation with BRAKER, and (3) protein clustering with OrthoFinder. A published long-read assembly of C. zeina (CMW25467) from Zambia was included and annotated using the same pipeline. This analysis revealed 790 non-shared accessory and 10,677 shared core orthogroups (genes) between the 31 isolates. Accessory gene content was largely shared between isolates from all countries, with a few genes unique to populations from Southern Africa (32) or East Africa (6). There was a significantly higher proportion of effector genes in the accessory secretome (44%) compared to the core secretome (24%). PCA, ADMIXTURE, and phylogenetic analysis using a neighbor-net network indicated a population structure with a geographical subdivision between the East African isolates and the Southern African isolates, although gene flow was also evident. The small pangenome and partial population differentiation indicated recent dispersal of C. zeina into Africa, possibly from 2 regional founder populations, followed by recurrent gene flow owing to widespread maize production across sub-Saharan Africa.

In silico secretome analyses of the polyphagous root-knot nematode Meloidogyne javanica: a resource for studying M. javanica secreted proteins.

BACKGROUND: Plant-parasitic nematodes (PPNs) that cause most damage include root-knot nematodes (RKNs) which are a major impediment to crop production. Root-knot nematodes, like other parasites, secrete proteins which are required for parasite proliferation and survival within the host during the infection process. RESULTS: Here, we used various computational tools to predict and identify classically and non-classically secreted proteins encoded in the Meloidogyne javanica genome. Furthermore, functional annotation analysis was performed using various integrated bioinformatic tools to determine the biological significance of the predicted secretome. In total, 7,458 proteins were identified as secreted ones. A large percentage of this secretome is comprised of small proteins of ≤ 300 aa sequence length. Functional analyses showed that M. javanica secretome comprises cell wall degrading enzymes for facilitating nematode invasion, and migration by disintegrating the complex plant cell wall components. In addition, peptidases and peptidase inhibitors are an important category of M. javanica secretome involved in compatible host-nematode interactions. CONCLUSION: This study identifies the putative secretome encoded in the M. javanica genome. Future experimental validation analyses can greatly benefit from this global analysis of M. javanica secretome. Equally, our analyses will advance knowledge of the interaction between plants and nematodes.

The genome of the king protea, Protea cynaroides.

The king protea (Protea cynaroides), an early-diverging eudicot, is the most iconic species from the Megadiverse Cape Floristic Region, and the national flower of South Africa. Perhaps best known for its iconic flower head, Protea is a key genus for the South African horticulture industry and cut-flower market. Ecologically, the genus and the family Proteaceae are important models for radiation and adaptation, particularly to soils with limited phosphorus bio-availability. Here, we present a high-quality chromosome-scale assembly of the P. cynaroides genome as the first representative of the fynbos biome. We reveal an ancestral whole-genome duplication event that occurred in the Proteaceae around the late Cretaceous that preceded the divergence of all crown groups within the family and its extant diversity in all Southern continents. The relatively stable genome structure of P. cynaroides is invaluable for comparative studies and for unveiling paleopolyploidy in other groups, such as the distantly related sister group Ranunculales. Comparative genomics in sequenced genomes of the Proteales shows loss of key arbuscular mycorrhizal symbiosis genes likely ancestral to the family, and possibly the order. The P. cynaroides genome empowers new research in plant diversification, horticulture and adaptation, particularly to nutrient-poor soils.

Haplotype mining panel for genetic dissection and breeding in Eucalyptus.

To improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic single-nucleotide polymorphism (SNP) markers. These markers fail to capture the large amount of haplotype diversity in these species, and thus multi-allelic markers are required. We aimed to develop a gene-based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6293 genes implicated in growth and wood properties, pest and disease resistance, and abiotic stress responses. We identified and phased 195 834 SNPs using a read-based phasing approach to reveal SNP-based haplotypes. A total of 8915 target regions (at 4637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13-4.52 haplotypes per target region in each species, and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species, and provide multi-allelic markers that can be used for genome-wide association studies and gene-based breeding approaches.

Calonectria queenslandica: Causal Agent of Eucalyptus Leaf Blight in Southern China.

Calonectria leaf blight caused by Calonectria spp. is among the most serious diseases affecting the health and sustainability of Eucalyptus plantations in southern China. Recent outbreaks of this disease in GuangDong Province prompted a need to identify the species involved. Typical symptoms of Calonectria leaf blight were observed on 2-year-old Eucalyptus urophylla × E. grandis trees in a plantation in the ZhaoQing region. In total, 38 Calonectria isolates were collected from 32 diseased trees. All isolates were identified using DNA sequence analyses of the translation elongation factor 1-α (tef1), ß-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) gene regions. Phylogenetic analyses revealed that Calonectria queenslandica was the dominant species, accounting for 81.6% of the isolates collected. Other species isolated included C. pseudoreteaudii (10.5%), C. reteaudii (5.3%), and C. aconidialis (2.6%). This is the first report of C. queenslandica in China and all isolates had identical sequences in all four gene regions. PCR amplification using primers targeting the MAT1-1-1 and MAT1-2-1 genes in all C. queenslandica isolates revealed that only the MAT1-2 idiomorph was present. The results suggest that C. queenslandica was introduced into the sampled area with very limited genetic diversity. Pathogenicity tests were conducted on two Eucalyptus genotypes widely planted in the GuangDong Province using isolates representing all species collected. The results showed that these species could all cause disease but the predominance of C. queenslandica on infected trees suggests that it is the major driver of the disease problem studied. Different Eucalyptus genotypes used in the pathogenicity tests differed in susceptibility to infection by the Calonectria spp. tested, providing opportunities to avoid leaf blight by deploying disease-tolerant planting stock.

Polyphasic evaluation and cytotoxic investigation of isolated cyanobacteria with an emphasis on potent activities of a Scytonema strain.

Cyanobacteria are phototrophic organisms widely found in most types of natural habitats in the tropical regions of the world. In this study, we isolated and identified cyanobacterial strains from paddy soil in Hanoi (Vietnam) and investigated their cytotoxic activities. Five isolated cyanobacterial strains showed distinctive profiles of gene sequences (rRNA 16S and rbcL), phylogenetic placements, and morphological characteristics. Based on the polyphasic evaluation, they were classified as Scytonema bilaspurense NK13, Hapalosiphon welwitschii MD2411, Aulosira sp. XN1103, Desikacharya sp. NS2000, and Desmonostoc sp. NK1813. The cytotoxic screening revealed that the extract of strain Scytonema bilaspurense NK13 exhibited potent cytotoxic activities against four human cell lines of HeLa cells, OVCAR-8 cells, HaCaT cells, and HEK-293T cells, with IC50 values of 3.8, 34.2, 21.6, and 0.6 µg/mL, respectively. This is the first time a well-classified Scytonema strain from tropical habitat in Southeast Asia has been recognized as a potential producer of cytotoxic compounds.

Diversity and Distribution of Calonectria Species from Plantation and Forest Soils in Fujian Province, China.

To meet the growing demand for wood and pulp products, Eucalyptus plantations have expanded rapidly during the past two decades, becoming an integral part of the southern China landscape. Leaf blight caused by various Calonectria spp., is a serious threat to these plantations. In order to explore the diversity and distribution of Calonectria spp. in Fujian Province soils, samples were collected in Eucalyptus plantations and adjacent plantings of Cunninghamia lanceolata, Phyllostachys heterocycle and Pinus massoniana as well as in natural forests. Three hundred and fifty-three Calonectria isolates were recovered from soil samples and they were identified based on a comparison of multilocus DNA sequence data for the act (actin), cmdA (calmodulin), his3 (histone H3), rpb2 (the second largest subunit of RNA polymerase), tef1 (translation elongation factor 1-alpha) and tub2 (ß-tubulin) gene regions, as well as morphological characteristics. Six known taxa including Calonectria aconidialis, Ca. hongkongensis, Ca. ilicicola, Ca. kyotensis, Ca. pacifica, Ca. pseudoreteaudii and one novel species described here as Ca. minensis sp. nov. were identified. Of these, Ca. aconidialis and Ca. kyotensis were the most prevalent species, and found in eight and seven sites, and four and five forest types, respectively. Calonectria spp. were most abundant in soils from Eucalyptus stands, followed by P. heterocycle and natural forests. Relatively few species were found in the soils associated with Cunninghamia lanceolata and Pinus massoniana. The abundance of known Calonectria spp. suggests that these fungi have been relatively well sampled in Fujian. The results are also consistent with the fact that most Calonectria diseases are found on Angiosperm as opposed to Gymnosperm plants.

Intra-Species Genomic Variation in the Pine Pathogen Fusarium circinatum.

Fusarium circinatum is an important global pathogen of pine trees. Genome plasticity has been observed in different isolates of the fungus, but no genome comparisons are available. To address this gap, we sequenced and assembled to chromosome level five isolates of F. circinatum. These genomes were analysed together with previously published genomes of F. circinatum isolates, FSP34 and KS17. Multi-sample variant calling identified a total of 461,683 micro variants (SNPs and small indels) and a total of 1828 macro structural variants of which 1717 were copy number variants and 111 were inversions. The variant density was higher on the sub-telomeric regions of chromosomes. Variant annotation revealed that genes involved in transcription, transport, metabolism and transmembrane proteins were overrepresented in gene sets that were affected by high impact variants. A core genome representing genomic elements that were conserved in all the isolates and a non-redundant pangenome representing all genomic elements is presented. Whole genome alignments showed that an average of 93% of the genomic elements were present in all isolates. The results of this study reveal that some genomic elements are not conserved within the isolates and some variants are high impact. The described genome-scale variations will help to inform novel disease management strategies against the pathogen.

Four New Species of Harringtonia: Unravelling the Laurel Wilt Fungal Genus.

Symbiosis between beetles and fungi arose multiple times during the evolution of both organisms. Some of the most biologically diverse and economically important are mutualisms in which the beetles cultivate and feed on fungi. Among these are bark beetles and Harringtonia, a fungal genus that produces Raffaelea-like asexual morph and hosts the causal agent of laurel wilt, H. lauricola (formerly Raffaelea lauricola). In this study, we propose four new species of Harringtonia associated with beetles from Belize and Florida (USA). We hope to contribute towards a more robust and inclusive phylogenetic framework for future studies on these beetle-fungi relationships and their potential impact in crops and forests worldwide.

Attitudes towards COVID-19 vaccination in patients with inflammatory bowel disease.

The majority of the Australian public are willing to have a Coronavirus disease 2019 (COVID-19) vaccination. It is unclear whether people with inflammatory bowel disease (IBD) have the same attitude towards COVID-19 vaccination. A survey was performed to assess the attitude of patients with IBD towards COVID-19 vaccination in South Australia. Two-thirds of surveyed patients with IBD were willing to accept COVID-19 vaccine. Females and younger patients were less likely to accept the COVID-19 vaccine, as were those who had never had a discussion around vaccines.

Molecular basis of cycloheximide resistance in the Ophiostomatales revealed.

Resistance to the antibiotic Cycloheximide has been reported for a number of fungal taxa. In particular, some yeasts are known to be highly resistant to this antibiotic. Early research showed that this resulted from a transition mutation in one of the 60S ribosomal protein genes. In addition to the yeasts, most genera and species in the Ophiostomatales are highly resistant to this antibiotic, which is widely used to selectively isolate these fungi. Whole-genome sequences are now available for numerous members of the Ophiostomatales providing an opportunity to determine whether the mechanism of resistance in these fungi is the same as that reported for yeast genera such as Kluyveromyces. We examined all the available genomes for the Ophiostomatales and discovered that a transition mutation in the gene coding for ribosomal protein eL42, which results in the substitution of the amino acid Proline to Glutamine, likely confers resistance to this antibiotic. This change across all genera in the Ophiostomatales suggests that the mutation arose early in the evolution of these fungi.

Population Genetic Analyses of Phytophthora cinnamomi Reveals Three Lineages and Movement Between Natural Vegetation and Avocado Orchards in South Africa.

Phytophthora cinnamomi is the causal agent of root rot, canker, and dieback of thousands of plant species around the globe. This oomycete not only causes severe economic losses to forestry and agricultural industries, but also threatens the health of various plants in natural ecosystems. In this study, 380 isolates of P. cinnamomi from four avocado production areas and two regions of natural vegetation in South Africa were investigated using 15 microsatellite markers. These populations were found to have a low level of genetic diversity and consisted of isolates from three lineages. Shared genotypes were detected between isolates from avocado orchards and natural vegetation, indicating the movement of isolates between these areas. The population from the Western Cape natural vegetation had the highest level of genotypic diversity and number of unique alleles, indicating this could be the point of introduction of P. cinnamomi to South Africa. Index of association analysis suggested that five of six populations were under linkage disequilibrium, suggesting a clonal mode of reproduction, whereas genotypes sampled from a recently established avocado orchard in the Western Cape were derived from a randomly recombining population. This study provided novel insights on the genetic diversity and spread of P. cinnamomi in South Africa. It also reported on the predominance of triploidy in natural occurring populations and provided evidence for recombination of P. cinnamomi for the first time. The presence of two dominant genotypes in all avocado production areas in South Africa highlight the importance of considering them in disease management and resistance breeding programs.

Haplogenome assembly reveals structural variation in Eucalyptus interspecific hybrids.

BACKGROUND: De novo phased (haplo)genome assembly using long-read DNA sequencing data has improved the detection and characterization of structural variants (SVs) in plant and animal genomes. Able to span across haplotypes, long reads allow phased, haplogenome assembly in highly outbred organisms such as forest trees. Eucalyptus tree species and interspecific hybrids are the most widely planted hardwood trees with F1 hybrids of Eucalyptus grandis and E. urophylla forming the bulk of fast-growing pulpwood plantations in subtropical regions. The extent of structural variation and its effect on interspecific hybridization is unknown in these trees. As a first step towards elucidating the extent of structural variation between the genomes of E. grandis and E. urophylla, we sequenced and assembled the haplogenomes contained in an F1 hybrid of the two species. FINDINGS: Using Nanopore sequencing and a trio-binning approach, we assembled the separate haplogenomes (566.7 Mb and 544.5 Mb) to 98.0% BUSCO completion. High-density SNP genetic linkage maps of both parents allowed scaffolding of 88.0% of the haplogenome contigs into 11 pseudo-chromosomes (scaffold N50 of 43.8 Mb and 42.5 Mb for the E. grandis and E. urophylla haplogenomes, respectively). We identify 48,729 SVs between the two haplogenomes providing the first detailed insight into genome structural rearrangement in these species. The two haplogenomes have similar gene content, 35,572 and 33,915 functionally annotated genes, of which 34.7% are contained in genome rearrangements. CONCLUSIONS: Knowledge of SV and haplotype diversity in the two species will form the basis for understanding the genetic basis of hybrid superiority in these trees.

The relevance of studying insect-nematode interactions for human disease.

Vertebrate-parasitic nematodes cause debilitating, chronic infections in millions of people worldwide. The burden of these so-called 'neglected tropical diseases' is often carried by poorer socioeconomic communities in part because research on parasitic nematodes and their vertebrate hosts is challenging and costly. However, complex biological and pathological processes can be modeled in simpler organisms. Here, we consider how insight into the interactions between entomopathogenic nematodes (EPN), their insect hosts and bacterial symbionts may reveal novel treatment targets for parasitic nematode infections. We argue that a combination of approaches that target nematodes, as well as the interaction of pathogens with insect vectors and bacterial symbionts, offer potentially effective, but underexplored opportunities.