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.

Chrysoporthe brasiliensis sp. nov. pathogenic to Melastomataceae in southeast Brazil.

Species in the Melastomataceae (Myrtales) include trees and woody shrubs that are amongst the most common hosts of Chrysoporthe and related fungi. These fungi cause stem cankers, branch death and in extreme cases, kill their hosts. Chrysoporthe-like fungi were observed on Miconia spp. and Rhynchanthera grandiflora (Melastomataceae) plants during tree disease surveys in south-eastern Brazil including the states of Minas Gerais and Rio de Janeiro. The aims of this study were to isolate and identify the fungi utilising morphological characteristics and phylogenetic analyses. This led to the identification of a new species of Chrysoporthe described here as Chrysoporthe brasilensis sp.nov. Inoculations were conducted on R. grandiflora and M. theaezans, showing that C. brasiliensis is an aggressive pathogen. This study adds to a growing number of reports of new and pathogenic species of Chrysoporthe that potentially threaten native Myrtales globally, including important trees such as Eucalyptus, both in natural ecosystems and in planted forests.

Unidirectional mating-type switching is underpinned by a conserved MAT1 locus architecture.

Unidirectional mating-type switching is a form of homothallic reproduction known only in a small number of filamentous ascomycetes. Their ascospores can give rise to either self-sterile isolates that require compatible partners for subsequent sexual reproduction, or self-fertile individuals capable of completing this process in isolation. The limited studies previously conducted in these fungi suggest that the differences in mating specificity are determined by the architecture of the MAT1 locus. In self-fertile isolates that have not undergone unidirectional mating-type switching, the locus contains both MAT1-1 and MAT1-2 mating-type genes, typical of primary homothallism. In the self-sterile isolates produced after a switching event, the MAT1-2 genes are lacking from the locus, likely due to a recombination-mediated deletion of the MAT1-2 gene information. To determine whether these arrangements of the MAT1 locus support unidirectional mating-type switching in the Ceratocystidaceae, the largest known fungal assemblage capable of this reproduction strategy, a combination of genetic and genomic approaches were used. The MAT1 locus was annotated in representative species of Ceratocystis, Endoconidiophora, and Davidsoniella. In all cases, MAT1-2 genes interrupted the MAT1-1-1 gene in self-fertile isolates. The MAT1-2 genes were flanked by two copies of a direct repeat that accurately predicted the boundaries of the deletion event that would yield the MAT1 locus of self-sterile isolates. Although the relative position of the MAT1-2 gene region differed among species, it always disrupted the MAT1-1-1 gene and/or its expression in the self-fertile MAT1 locus. Following switching, this gene and/or its expression was restored in the self-sterile arrangement of the locus. This mirrors what has been reported in other species capable of unidirectional mating-type switching, providing the strongest support for a conserved MAT1 locus structure that is associated with this process. This study contributes to our understanding of the evolution of unidirectional mating-type switching.

Needles in fungal haystacks: Discovery of a putative a-factor pheromone and a unique mating strategy in the Leotiomycetes.

The Leotiomycetes is a hugely diverse group of fungi, accommodating a wide variety of important plant and animal pathogens, ericoid mycorrhizal fungi, as well as producers of antibiotics. Despite their importance, the genetics of these fungi remain relatively understudied, particularly as they don't include model taxa. For example, sexual reproduction and the genetic mechanisms that underly this process are poorly understood in the Leotiomycetes. We exploited publicly available genomic and transcriptomic resources to identify genes of the mating-type locus and pheromone response pathway in an effort to characterize the mating strategies and behaviors of 124 Leotiomycete species. Our analyses identified a putative a-factor mating pheromone in these species. This significant finding represents the first identification of this gene in Pezizomycotina species outside of the Sordariomycetes. A unique mating strategy was also discovered in Lachnellula species that appear to have lost the need for the primary MAT1-1-1 protein. Ancestral state reconstruction enabled the identification of numerous transitions between homothallism and heterothallism in the Leotiomycetes and suggests a heterothallic ancestor for this group. This comprehensive catalog of mating-related genes from such a large group of fungi provides a rich resource from which in-depth, functional studies can be conducted in these economically and ecologically important species.

New Resinogalea species from Araucaria araucana resin in Chile and reclassification of the genus in the Cryptocaliciomycetidae.

Araucaria araucana is an ancient conifer, native to the mountain ranges in Chile and Argentina. These trees host a large number of organisms, mainly insects, strongly or even exclusively associated with them. The recent emergence of a novel canker disease on A. araucana has emphasised the importance of fungi associated with these iconic trees and has resulted in the discovery of various new species. In this study, we considered the identity of an unknown calicioid fungus consistently found on resin on the branches of A. araucana. Preliminary phylogenetic analyses placed isolates in the recently described sub-class Cryptocaliciomycetidae, closest to Cryptocalicium blascoi. However, the morphology of the ascomata and its occurrence in a unique niche suggested that the closest relative could be Resinogalea humboldtensis (Bruceomycetaceae, incertae sedis), a fungus with similar sporing structures found on resin of Araucaria humboldtensis in New Caledonia. There are no living cultures or sequence data available for either R. humboldtensis or its supposed closest relative, Bruceomyces castoris, precluding sequence-based comparisons. Morphological comparisons of the sporing structures on A. araucana confirmed that the ascomatal morphology of our unknown calicioid fungus and R. humboldtensis are almost identical and resemble each other more so than B. castoris or Cr. blascoi. A phylogenetic analysis based on the small subunit (SSU), internal transcribed spacer (ITS) and large subunit (LSU) rDNA regions resolved our strains into two clades with Cr. blascoi as its closest relative. Further analyses applying the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) based on ITS, mini chromosome maintenance protein complex (MCM7), RNA polymerase II second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF) gene regions, confirmed that strains represent two new species. Based on our morphological observations and phylogenetic analyses, we introduce two new Resinogalea species, R. araucana and R. tapulicola, and reclassify the genus in the subclass Cryptocaliciomycetidae.

The Global Forest Health Crisis: A Public-Good Social Dilemma in Need of International Collective Action.

Society is confronted by interconnected threats to ecological sustainability. Among these is the devastation of forests by destructive non-native pathogens and insects introduced through global trade, leading to the loss of critical ecosystem services and a global forest health crisis. We argue that the forest health crisis is a public-good social dilemma and propose a response framework that incorporates principles of collective action. This framework enables scientists to better engage policymakers and empowers the public to advocate for proactive biosecurity and forest health management. Collective action in forest health features broadly inclusive stakeholder engagement to build trust and set goals; accountability for destructive pest introductions; pooled support for weakest-link partners; and inclusion of intrinsic and nonmarket values of forest ecosystems in risk assessment. We provide short-term and longer-term measures that incorporate the above principles to shift the societal and ecological forest health paradigm to a more resilient state.

Structure and number of mating pheromone genes is closely linked to sexual reproductive strategy in Huntiella.

BACKGROUND: Huntiella resides in the Ceratocystidaceae, a family of fungi that accommodates important plant pathogens and insect-associated saprotrophs. Species in the genus have either heterothallic or unisexual (a form of homothallism) mating systems, providing an opportunity to investigate the genetic mechanisms that enable transitions between reproductive strategies in related species. Two newly sequenced Huntiella genomes are introduced in this study and comparative genomics and transcriptomics tools are used to investigate the differences between heterothallism and unisexuality across the genus. RESULTS: Heterothallic species harbored up to seven copies of the a-factor pheromone, each of which possessed numerous mature peptide repeats. In comparison, unisexual Huntiella species had only two or three copies of this gene, each with fewer repeats. Similarly, while the heterothallic species expressed up to 12 copies of the mature α-factor pheromone, unisexual species had up to six copies. These significant differences imply that unisexual Huntiella species do not rely on a mating partner recognition system in the same way that heterothallic fungi do. CONCLUSION: While it is suspected that mating type-independent pheromone expression is the mechanism allowing for unisexual reproduction in Huntiella species, our results suggest that the transition to unisexuality may also have been associated with changes in the genes governing the pheromone pathway. While these results are specifically related to Huntiella, they provide clues leading to a better understanding of sexual reproduction and the fluidity of mating strategies in fungi more broadly.

Efficacy of Commercially Available Entomopathogenic Agents against the Polyphagous Shot Hole Borer in South Africa.

The invasive ambrosia beetle, Euwallacea fornicatus, was first reported in South Africa in 2018. The beetle has now spread to eight provinces of the country and has had a devastating impact on both native and non-native tree species. This is especially true for trees located in urban and peri-urban environments. Recent predictions are that the South African E. fornicatus invasion will cost an estimated ZAR 275 billion (approx. USD 16 billion) if it continues to spread uncontrollably, justifying an urgent need for its effective management in the country. One option is biological control, which is preferred over the use of chemicals due to its lower environmental impact. We tested two broad-spectrum fungal entomopathogenic agents, Eco-Bb® and Bio-Insek, which are commercially available in South Africa, for efficacy against E. fornicatus. Initial laboratory assays yielded promising results. However, beetle infestation trials using treated pieces of woody castor bean stems showed little effect on beetle survival and reproduction.

Population structure and diversity of the needle pathogen Dothistroma pini suggests human-mediated movement in Europe.

Dothistroma needle blight (DNB) is an important disease of Pinus species that can be caused by one of two distinct but closely related pathogens; Dothistroma septosporum and Dothistroma pini. Dothistroma septosporum has a wide geographic distribution and is relatively well-known. In contrast, D. pini is known only from the United States and Europe, and there is a distinct lack of knowledge regarding its population structure and genetic diversity. The recent development of 16 microsatellite markers for D. pini provided an opportunity to investigate the diversity, structure, and mode of reproduction for populations collected over a period of 12 years, on eight different hosts in Europe. In total, 345 isolates from Belgium, the Czech Republic, France, Hungary, Romania, Western Russia, Serbia, Slovakia, Slovenia, Spain, Switzerland, and Ukraine were screened using microsatellite and species-specific mating type markers. A total of 109 unique multilocus haplotypes were identified and structure analyses suggested that the populations are influenced by location rather than host species. Populations from France and Spain displayed the highest levels of genetic diversity followed by the population in Ukraine. Both mating types were detected in most countries, with the exception of Hungary, Russia and Slovenia. Evidence for sexual recombination was supported only in the population from Spain. The observed population structure and several shared haplotypes between non-bordering countries provides good evidence that the movement of D. pini in Europe has been strongly influenced by human activity in Europe.

A LAMP Assay for Rapid Detection of the Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen Fusarium circinatum is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by F. circinatum can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to F. circinatum. Using a globally representative collection of F. circinatum isolates and other closely related species, we have demonstrated that the assay can be used to identify F. circinatum across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.

Five new Graphium species from hardwood trees in Poland.

Graphium species form a well-supported monophyletic lineage within the Microascales (Ascomycota). Members of this genus can be found in association with bark beetles, as well as on tree wounds and in soils. During surveys of bark and ambrosia beetle-associated fungi and cavities made by woodpeckers on hardwood trees in Poland, many isolates with an affinity to Graphium were recovered. They were identified based on their morphological characters and sequence data for the internal transcribed spacer (ITS), 28S rDNA, ß-tubulin (TUB2), and translation elongation factor 1-α (TEF1) gene regions. The results revealed five new species, described here as G. brachiatum, G. longistipitatum, G. polonicum, G. radicatum, and G. trypophloei.

A Robust Disease Scoring Method to Screen Eucalyptus for Resistance Against the Aggressive Leaf Pathogen Teratosphaeria destructans.

Shoot and leaf blight caused by Teratosphaeria destructans is one of the most devastating foliar diseases on Eucalyptus. Therefore, breeding for resistance to this disease is considered urgent. Differences in susceptibility to T. destructans have been observed in the field but a robust inoculation protocol has, until recently, been unavailable and a disease scoring method for precise phenotyping has not been established. A first objective of this study was to determine the optimal conidial concentration for T. destructans inoculations on a susceptible Eucalyptus host. This concentration was then used to determine differences in susceptibility of six genotypes of Eucalyptus grandis × E. urophylla to the pathogen by assessing the percentage of infected stomata using electron microscopy and the percentage of leaf area covered by lesions (PLACL) using image processing. In addition, we developed a disease susceptibility index (SI) of six categories ranging from highly resistant (SI = 0) to highly susceptible (SI = 1.5 to 2). The more resistant genotypes were moderately resistant, with an SI value of 0.49 to 0.54 and a PLACL of 6.5 to 9%. In contrast, the more susceptible genotype scored an SI of 1.52 and PLACL of 48%. Host susceptibility was also assessed relative to the sporulation of the pathogen. This showed that the percentage of sporulation was not significantly correlated with host resistance. Overall, the results provide the basis for rigorous screening and selection of resistant genotypes to the disease caused by T. destructans using artificial inoculation.

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.

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.

Comparison of Hyphal Fragments and Spores to Evaluate the Pathogenicity of the Eucalyptus Leaf and Shoot Pathogen Calonectria pseudoreteaudii.

The genus Calonectria includes many aggressive plant-pathogenic species with a worldwide distribution. Calonectria leaf blight is one of the most prominent diseases of Eucalyptus trees in Southeast Asian and South American plantations. Inoculation trials to evaluate pathogenicity of Calonectria spp. typically use conidial suspensions but this is not possible for species that do not sporulate sufficiently in culture. Calonectria pseudoreteaudii is one of the species that is most aggressive to Eucalyptus in China but most isolates fail to produce conidia in culture, requiring an alternative procedure for artificial inoculation. This study compared inoculations utilizing conidial and hyphal fragment suspensions. Two Eucalyptus genotypes were used, and these were inoculated with different concentrations of hyphal fragments or conidia of three C. pseudoreteaudii isolates. Three days after inoculation, the treated Eucalyptus plants displayed similar disease symptoms, regardless of whether they had been inoculated with conidia or hyphal fragments. This was consistent for all C. pseudoreteaudii isolates and also the different Eucalyptus genotypes. The results demonstrate that hyphal fragment suspensions can be used to provide a reliable indication of C. pseudoreteaudii isolate pathogenicity when conidia are not available for inoculation studies.

Novel mating-type-associated genes and gene fragments in the genomes of Mycosphaerellaceae and Teratosphaeriaceae fungi.

The mating-type (MAT1) locus encodes transcription factors essential for the onset of the sexual cycle in ascomycete fungi. This locus has been characterised in only a few heterothallic, plant pathogenic Mycosphaerellaceae and Teratosphaeriaceae. We used available genome sequences for Mycosphaerellales species to investigate the presence of two unique mating-type-associated features. The accessory MAT1 genes, MAT1-1-10 (MATORF2) and MAT1-2-12 (MATORF1), typically occurred in both MAT idiomorphs of Mycosphaerellaceae species. In contrast, they were associated with only one idiomorph in Teratosphaeriaceae species. In Pseudocercospora, phylogenetic analyses showed that homologs present in different idiomorphs were paralogous and subject to different selective pressures, indicating that their evolution is linked to mating type. In almost half of the investigated Mycosphaerellales genomes, numerous short fragment sequences, almost identical to portions of the MAT1-1-1 and MAT1-2-1 genes, were present in multiple areas outside of the MAT1 locus. Aligned to the MAT1 genes, these sequences resembled an mRNA transcript. Fragment sequences were similar among species groups and occurred at the same genomic positions, implying that monophyletic groups have the same origins of these sequences. Although the functions of the MAT fragment sequences and accessory MAT1 genes remain unknown, both were expressed in the representative Mycosphaerellaceae and Teratosphaeriaceae species that were investigated.

Calonectria in the age of genes and genomes: Towards understanding an important but relatively unknown group of pathogens.

The genus Calonectria includes many aggressive plant pathogens causing diseases on various agricultural crops as well as forestry and ornamental tree species. Some species have been accidentally introduced into new environments via international trade of putatively asymptomatic plant germplasm or contaminated soil, resulting in significant economic losses. This review provides an overview of the taxonomy, population biology, and pathology of Calonectria species, specifically emerging from contemporary studies that have relied on DNA-based technologies. The growing importance of genomics in future research is highlighted. A life cycle is proposed for Calonectria species, aimed at improving our ability to manage diseases caused by these pathogens.