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.

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.

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.

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.

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.

Genome of the destructive oomycete Phytophthora cinnamomi provides insights into its pathogenicity and adaptive potential.

BACKGROUND: Phytophthora cinnamomi is an oomycete pathogen of global relevance. It is considered as one of the most invasive species, which has caused irreversible damage to natural ecosystems and horticultural crops. There is currently a lack of a high-quality reference genome for this species despite several attempts that have been made towards sequencing its genome. The lack of a good quality genome sequence has been a setback for various genetic and genomic research to be done on this species. As a consequence, little is known regarding its genome characteristics and how these contribute to its pathogenicity and invasiveness. RESULTS: In this work we generated a high-quality genome sequence and annotation for P. cinnamomi using a combination of Oxford Nanopore and Illumina sequencing technologies. The annotation was done using RNA-Seq data as supporting gene evidence. The final assembly consisted of 133 scaffolds, with an estimated genome size of 109.7 Mb, N50 of 1.18 Mb, and BUSCO completeness score of 97.5%. Genome partitioning analysis revealed that P. cinnamomi has a two-speed genome characteristic, similar to that of other oomycetes and fungal plant pathogens. In planta gene expression analysis revealed up-regulation of pathogenicity-related genes, suggesting their important roles during infection and host degradation. CONCLUSION: This study has provided a high-quality reference genome and annotation for P. cinnamomi. This is among the best assembled genomes for any Phytophthora species assembled to date and thus resulted in improved identification and characterization of pathogenicity-related genes, some of which were undetected in previous versions of genome assemblies. Phytophthora cinnamomi harbours a large number of effector genes which are located in the gene-poor regions of the genome. This unique genomic partitioning provides P. cinnamomi with a high level of adaptability and could contribute to its success as a highly invasive species. Finally, the genome sequence, its annotation and the pathogenicity effectors identified in this study will serve as an important resource that will enable future studies to better understand and mitigate the impact of this important pathogen.

Ophiostomatalean fungi associated with wood boring beetles in South Africa including two new species.

Ambrosia beetles are small wood inhabiting members of the Curculionidae that have evolved obligate symbioses with fungi. The fungal symbionts concentrate nutrients from within infested trees into a usable form for their beetle partners, which then utilize the fungi as their primary source of nutrition. Ambrosia beetle species associate with one or more primary symbiotic fungal species, but they also vector auxiliary symbionts, which may provide the beetle with developmental or ecological advantages. In this study we isolated and identified ophiostomatalean fungi associated with ambrosia beetles occurring in a native forest area in South Africa. Using a modified Bambara beetle trap, living ambrosia beetle specimens were collected and their fungal symbionts isolated. Four beetle species, three Scolytinae and one Bostrichidae, were collected. Five species of ophiostomatalean fungi were isolated from the beetles and were identified using both morphological characters and DNA sequence data. One of these species, Raffaelea sulphurea, was recorded from South Africa for the first time and two novel species were described as Ceratocystiopsis lunata sp. nov. and Raffaelea promiscua sp. nov.

Genome comparisons suggest an association between Ceratocystis host adaptations and effector clusters in unique transposable element families.

Ceratocystis fimbriata is a host specific fungal pathogen of sweet potato (Ipomoea batatas). The closely related species, C. manginecans, is an important pathogen of trees (e.g. Acacia mangium and Mangifera indica) but has never been isolated from tuber crops. The genetic factors that determine the host range and host specificity of these species have not been determined. The aim of this study was to compare the genomes of C. fimbriata and C. manginecans in order to identify species-specific genetic differences that could be associated with host specificity. This included whole-genome alignments as well as comparisons of gene content and transposable elements (TEs). The genomes of the two species were found to be very similar, sharing similar catalogues of CAZymes, peptidases and lipases. However, the genomes of the two species also varied, harbouring species-specific genes (e.g. small secreted effectors, nutrient processing proteins and stress response proteins). A portion of the TEs identified (17%) had a unique distribution in each species. Transposable elements appeared to have played a prominent role in the divergence of the two species because they were strongly associated with chromosomal translocations and inversions as well as with unique genomic regions containing species-specific genes. Two large effector clusters, with unique TEs in each species, were identified. These effectors displayed non-synonymous mutations and deletions, conserved within a species, and could serve as mutational hot-spots for the development of host specificity in the two species.

Mating strategy and mating type distribution in six global populations of the Eucalyptus foliar pathogen Teratosphaeria destructans.

Teratosphaeria destructans is an aggressive fungal pathogen causing leaf and shoot blight on young Eucalyptus trees in plantations. The disease occurs across tropical and subtropical regions of South East Asia and has recently been found in South Africa. Asexual structures of the pathogen are produced on infected tissues, but sexual structures have never been observed. The aim of this study was to investigate the reproductive biology of T. destructans by characterising its mating type (MAT1) locus and investigating its potential for sexual recombination. We found that T. destructans has a heterothallic mating system, with either the MAT1-1-1 and MAT1-1-10 genes (MAT1-1 idiomorph) or the MAT1-2-1 and MAT1-2-12 genes (MAT1-2 idiomorph) present in a single individual. With a multiplex PCR assay, it was possible to distinguish the two MAT idiomorphs in several Teratosphaeria species and this approach was applied to six global populations of T. destructans. Although both mating types occurred in the South East Asian populations, a single mating type dominated each population. Isolates from the recent disease outbreak in South Africa comprised only a single mating type. Attempts to induce a sexual cycle in vitro using strains of opposite mating type were not successful. The uneven distribution of mating types in populations of T. destructans and the presence of only an asexual state on infected tissues suggests the absence of or at least a minor role for sexual reproduction where the pathogen occurs on non-native Eucalyptus in plantations.

Wounds on Rapanea melanophloeos provide habitat for a large diversity of Ophiostomatales including four new species.

Rapanea melanophloeos, an important canopy tree in Afromontane forests, is commonly utilised for medicinal bark harvesting. Wounds created from these activities provide entrance for many fungi, including arthropod-associated members of the Ophiostomatales and Microascales (ophiostomatoid fungi). In this study we assessed the diversity of wound-associated Ophiostomatales on storm-damaged R. melanophloeos trees in the Afromontane forests of South Africa. Five species were identified based on micro-morphological and molecular phylogenetic analyses. These included Ophiostoma stenoceras and four newly described taxa Sporothrix itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov. and O. noisomeae sp. nov. Four of these are members of the S. schenckii-O. stenoceras complex (O. stenoceras, S. itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov.) while O. noisomeae groups basal in the Ophiostomatales alongside the S. lignivora complex and Graphilbum. In addition to other taxa known from this host, the present study shows that there is a rich, yet still poorly explored, diversity of Ophiostomatales associated with R. melanophloeos in Afromontane forests. More taxa are likely to be discovered with increased research effort. These must be assessed in terms of pathogenicity towards this ecologically and economically important tree.

New species of Ophiostomatales from Scolytinae and Platypodinae beetles in the Cape Floristic Region, including the discovery of the sexual state of Raffaelea.

Olea capensis and Rapanea melanophloeos are important canopy trees in South African Afromontane forests. Dying or recently dead individuals of these trees are often infested by Scolytinae and Platypodinae (Curculionidae) beetles. Fungi were isolated from the surfaces of beetles emerging from wood samples and their galleries. Based on micro-morphological and phylogenetic analyses, four fungal species in the Ophiostomatales were isolated. These were Sporothrix pallida and three taxa here newly described as Sporothrix aemulophila sp. nov., Raffaelea vaginata sp. nov. and Raffaelea rapaneae sp. nov. This study represents the first collection of S. pallida, a species known from many environmental samples from across the world, from Scolytinae beetles. S. aemulophila sp. nov. is an associate of the ambrosia beetle Xyleborinus aemulus. R. rapaneae sp. nov. and R. vaginata sp. nov. were associated with a Lanurgus sp. and Platypodinae beetle, respectively, and represent the first Raffaelea spp. reported from the Cape Floristic Region. Of significance is that R. vaginata produced a sexual state analogous with those of Ophiostoma seticolle and O. deltoideosporum that also grouped in our analyses in Raffaelea s. str., to date considered an asexual genus. The morphology of the ossiform ascospores and anamorphs of the three species corresponded and the generic circumscription of Raffaelea is thus emended to accommodate sexual states. The two known species are provided with new combinations, namely Raffaelea seticollis (R.W. Davidson) Z.W. de Beer and T.A. Duong comb. nov. and Raffaelea deltoideospora (Olchow. and J. Reid) Z.W. de Beer and T.A. Duong comb. nov.

Taxonomy and phylogeny of the Leptographium procerum complex, including Leptographium sinense sp. nov. and Leptographium longiconidiophorum sp. nov.

Leptographium procerum (Ophiostomatales, Ascomycota) is a well-known fungal associate of pine root-infesting bark beetles and weevils, occurring in several countries of the world. The fungus is not a primary pathogen but has been associated with white pine root decline in the USA and with serious damage caused by the introduced red turpentine beetle (RTB) Dendroctonus valens in China. Several species closely related to L. procerum have been described during the past decade. The aim of this study was to reevaluate species boundaries in the L. procerum complex using multigene phylogenetic analyses and morphological comparisons. Phylogenetic analyses of seven gene regions (ITS2-LSU, actin, ß-tubulin, calmodulin, translation elongation factor 1-α, and the mating type genes MAT1-1-3 and MAT1-2-1) distinguished between nine species in the complex. These included L. procerum, L. bhutanense, L. gracile, L. profanum, L. pini-densiflorae, L. sibiricum, L. sinoprocerum, as well as two new species described here as Leptographium sinense sp. nov. from Hylobitelus xiaoi on Pinus elliottii in China, and Leptographium longiconidiophorum sp. nov. from Pinus densiflora in Japan. Leptographium latens is reduced to synonymy with L. gracile, and an epitype is designated for L. procerum, because a living culture associated with the holotype of L. procerum did not exist. Amplification patterns of the mating type genes suggest that all known species in the L. procerum complex are heterothallic, although sexual states have not been observed for any of the species. The results also suggest that Eastern Asia is most probably the centre of species diversity for the L. procerum complex.

MAT gene idiomorphs suggest a heterothallic sexual cycle in a predominantly asexual and important pine pathogen.

Diplodia pinea (=Sphaeropsis sapinea) is a well-known and economically important latent pathogen of Pinus spp. in many parts of the world. Despite intensive scrutiny, its sexual state has never been observed and the fungus has thus been considered exclusively asexual. It was, therefore, surprising that a recent population genetics study showed high genotypic diversity and random association of alleles in a number of populations, suggesting that the pathogen has a cryptic sexual stage. Using the genome sequence of two individual D. pinea isolates, we interrogated the structure of the MAT locus in this fungus. The results suggested that D. pinea is heterothallic (self-sterile) with complete and apparently functional copies of the MAT genes containing the α-1 and HMG domains present in different isolates. In addition to the MAT1-2-1 and MAT1-1-1 genes, we found a MAT1-1-4 gene in the MAT1-1 idiomorph and a novel MAT1-2-5 gene in the MAT1-2 idiomorph. Importantly, the frequencies of occurrence of both idiomorphs in populations examined were not significantly different from a 1:1 ratio, which would be expected in sexually reproducing populations. Although the sexual state has never been observed, the results strongly suggest that D. pinea has a cryptic, heterothallic sexual cycle.

Multigene phylogenies of Ophiostomataceae associated with Monterey pine bark beetles in Spain reveal three new fungal species.

Ophiostoma species, some of which cause sapstain in timber and/or are mild pathogens, are common fungal associates of bark beetles (Coleoptera: Scolytinae). Three new Ophiostomataceae from Spain are recognized in the present study based on comparisons of sequence data for three gene regions as well as morphological characteristics. The new taxa are described as Ophiostoma nebulare sp. nov., Ophiostoma euskadiense sp. nov. and Graphilbum crescericum sp. nov.

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.

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.

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.