Predicting interactions of the frass-associated yeast Hyphopichia heimii with Olea europaea subsp. cuspidata and twig-boring bark beetles.

Bark beetles are destructive insect pests known to form symbioses with different fungal taxa, including yeasts. The aim of this study was to (1) determine the prevalence of the rare yeast Hyphopichia heimii in bark beetle frass from wild olive trees in South Africa and to (2) predict the potential interaction of this yeast with trees and bark beetles. Twenty-eight culturable yeast species were isolated from frass in 35 bark beetle galleries, including representatives of H. heimii from nine samples. Physiological characterization of H. heimii isolates revealed that none was able to degrade complex polymers present in hemicellulose; however, all were able to assimilate sucrose and cellobiose, sugars associated with an arboreal habitat. All isolates were able to produce the auxin indole acetic acid, indicative of a potential symbiosis with the tree. Sterol analysis revealed that the isolates possessed ergosterol quantities ranging from 3.644 ± 0.119 to 13.920 ± 1.230 mg/g dry cell weight, which suggested that H. heimii could serve as a source of sterols in bark beetle diets, as is known for other bark beetle-associated fungi. In addition, gas chromatography-mass spectrometry demonstrated that at least one of the isolates, Hyphopichia heimii CAB 1614, was able to convert the insect pheromone cis-verbenol to the anti-aggregation pheromone verbenone. This indicated that H. heimii could potentially influence beetle behaviour. These results support the contention of a tripartite symbiosis between H. heimii, olive trees, and bark beetles.

Persistence of ecologically similar fungi in a restricted floral niche.

Fungi in the genera Knoxdaviesia and Sporothrix dominate fungal communities within Protea flowerheads and seed cones (infructescences). Despite apparently similar ecologies, they show strong host recurrence and often occupy the same individual infructescence. Differences in host chemistry explain their host consistency, but the factors that allow co-occupancy of multiple species within individual infructescences are unknown. Sporothrix splendens and K. proteae often grow on different senescent tissue types within infructescences of their P. repens host, indicating that substrate-related differences aid their co-occupancy. Sporothrix phasma and K. capensis grow on the same tissues of P. neriifolia suggesting neutral competitive abilities. Here we test the hypothesis that differences in host-tissues dictate competitive abilities of these fungi and explain their co-occupancy of this spatially restricted niche. Media were prepared from infructescence bases, bracts, seeds, or pollen presenters of P. neriifolia and P. repens. As expected, K. capensis was unable to grow on seeds whilst S. phasma could. As hypothesised, K. capensis and S. phasma had equal competitive abilities on pollen presenters, appearing to explain their co-occupancy of this resource. Growth of K. proteae was significantly enhanced on pollen presenters while that of S. splendens was the same as the control. Knoxdavesia proteae grew significantly faster than S. splendens on all tissue types. Despite this, S. splendens was a superior competitor on all tissue types. For K. proteae to co-occupy infructescences with S. splendens for extended periods, it likely needs to colonize pollen presenters before the arrival of S. splendens.

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.

Early colonization of Protea flowers enable dominance of competitively weak saprobic fungi in seed cones, benefitting their hosts.

Sporothrix and Knoxdaviesia fungi use pollinators to colonize Protea flowers at anthesis. These saprobes remain dominant in the nutrient-rich, fire-retardant Protea seed-cones (infructescences) for at least a year after flowering. We tested the hypothesis that they competitively exclude potentially detrimental fungi from infructescences during this time. We compared seed set and longevity of infructescences containing Sporothrix and Knoxdaviesia vs. those that contain 'contaminant' saprobes. Hereafter we evaluated their competitive abilities against the 'contaminant' saprobes. Infructescences devoid of Sporothrix and Knoxdaviesia were dominated by Penicillium cf. toxicarium, Cladosporium cf. cladosporoides and Fusarium cf. anthophilum. Sporothrix and Knoxdaviesia presence did not affect seed viability, but infructescences persisted longer than those colonised by 'contaminant' fungi. The 'contaminant' species were stronger competitors than Sporothrix and Knoxdaviesia. However, Sporothrix and Knoxdaviesia could defend captured space well against 'contaminant' species. This effect was enhanced when fungal taxa grew on media prepared from their usual Protea host species, clarifying their dominance and host consistency observed in the field. Sporothrix and Knoxdaviesia from Protea are therefore weak competitors against common saprobes, especially when growing on alternative hosts, and need to colonise flowers very early (before colonization by other fungi) to dominate in this environment. They may delay seed release from infructescences longer than if these are colonised by other saprobes, increasing chances of seed release to occur after fire, when conditions are more favourable for Protea recruitment.

A high-quality fungal genome assembly resolved from a sample accidentally contaminated by multiple taxa.

Contamination in sequenced genomes is a relatively common problem and several methods to remove non-target sequences have been devised. Typically, the target and contaminating organisms reside in different kingdoms, simplifying their separation. The authors present the case of a genome for the ascomycete fungus Teratosphaeria eucalypti, contaminated by another ascomycete fungus and a bacterium. Approaching the problem as a low-complexity metagenomics project, the authors used two available software programs, BlobToolKit and anvi'o, to filter the contaminated genome. Both the de novo and reference-assisted approaches yielded a high-quality draft genome assembly for the target fungus. Incorporating reference sequences increased assembly completeness and visualization elucidated previously unknown genome features. The authors suggest that visualization should be routine in any sequencing project, regardless of suspected contamination.

Fire impacts bacterial composition in Protea repens (Proteaceae) infructescences.

The diverse bacterial communities in and around plants provide important benefits, such as protection against pathogens and cycling of essential minerals through decomposition of moribund plant biomass. Biodiverse fynbos landscapes generally have limited deadwood habitats due to the absence of large trees and frequent fire. In this study, we determined the effect of a fire disturbance on the bacterial communities in a fynbos landscape dominated by the shrub Protea repens using 16S ribosomal RNA amplicon sequencing. The bacterial community composition in newly formed fruiting structures (infructescences) and soil at a recently burnt site was different from that in an unburnt site. Bacteria inhabiting P. repens infructescences were similar to well-known taxa from decomposing wood and litter. This suggests a putative role for these aboveground plant structures as reservoirs for postfire decomposer bacteria. The results imply that inordinately frequent fires, which are commonplace in the Anthropocene, are a significant disturbance to bacterial communities and could affect the diversity of potentially important microbes from these landscapes.

Genetic response to nitrogen starvation in the aggressive Eucalyptus foliar pathogen Teratosphaeria destructans.

Teratosphaeria destructans is one of the most aggressive foliar pathogens of Eucalyptus. The biological factors underpinning T. destructans infections, which include shoot and leaf blight on young trees, have never been interrogated. Thus, the means by which the pathogen modifies its host environment to overcome host defences remain unknown. By applying transcriptome sequencing, the aim of this study was to compare gene expression in a South African isolate of T. destructans grown on nitrogen-deficient and complete media. This made it possible to identify upregulated genes in a nitrogen-starved environment, often linked to the pathogenicity of the fungus. The results support the hypothesis that nitrogen starvation in T. destructans likely mirrors an in planta genetic response. This is because 45% of genes that were highly upregulated under nitrogen starvation have previously been reported to be associated with infection in other pathogen systems. These included several CAZymes, fungal effector proteins, peptidases, kinases, toxins, lipases and proteins associated with detoxification of toxic compounds. Twenty-five secondary metabolites were identified and expressed in both nitrogen-deficient and complete conditions. Additionally, the most highly expressed genes in both growth conditions had pathogenicity-related functions. This study highlights the large number of expressed genes associated with pathogenicity and overcoming plant defences. As such, the generated baseline knowledge regarding pathogenicity and aggressiveness in T. destructans is a valuable reference for future in planta work.

Tree canopy arthropods have idiosyncratic responses to plant ecophysiological traits in a warm temperate forest complex.

Biodiversity studies on forest canopies often have narrow arthropod taxonomic focus, or refer to a single species of tree. In response, and to better understand the wide range of drivers of arthropod diversity in tree canopies, we conducted a large-scale, multi-taxon study which (a) included effect of immediate surroundings of an individual tree on plant physiological features, and (b), how these features affect compositional and functional arthropod diversity, in a warm, southern Afro-temperate forest. We found that tree species differed significantly in plant physiological features and arthropod diversity patterns. Surprisingly, we found negative correlation between surrounding canopy cover, and both foliar carbon and arthropod diversity in host trees, regardless of tree species. Subtle, tree intraspecific variation in physiological features correlated significantly with arthropod diversity measures, but direction and strength of correlations differed among tree species. These findings illustrate great complexity in how canopy arthropods respond to specific tree species, to immediate surroundings of host trees, and to tree physiological features. We conclude that in natural forests, loss of even one tree species, as well as homogenization of the crown layer and/or human-induced environmental change, could lead to profound and unpredictable canopy arthropod biodiversity responses, threatening forest integrity.

Interplay between differential competition and actions of spore-vectors explain host exclusivity of saprobic fungi in Protea flowers.

Protea flowers host saprobic Knoxdaviesia and Sporothrix fungi that are dispersed by pollinating insects and birds. Different Protea species contain sympatric populations of different fungal species. For example, P. repens host S. splendens and K. proteae, while P. neriifolia host K. capensis and S. phasma. Even though all fungi can grow vigorously on alternative hosts and they share the same spore vector species, they rarely colonise alternative hosts. We investigated the role of fungal differential competitive abilities on their usual and alternative hosts to explain their host exclusivity. In a de Wit replacement series experiment, S. splendens outcompeted and later overgrew all other fungi on media prepared from its usual and alternative hosts. Host exclusivity of S. splendens on P. repens may therefore be maintained by restricted movement of spore vectors rather than weaker competitive abilities on alternative hosts. On their preferred hosts, S. splendens and S. phasma rapidly overgrew Knoxdavesia species with which they do not usually share a host, explaining host exclusivity of the Knoxdavesia species. Knoxdaviesia proteae likely only persist on P. repens with sympatric S. splendens if it colonizes flowers earlier, in a different area, or completes its life cycle before being overgrown. On their usual P. neriifolia host, K. capensis and S. phasma had neutralistic interactions and S. phasma could not overgrow K. capensis, explaining their co-existence. Host exclusivity of saprobic Protea-associated Knoxdaviesia and Sporothrix may therefore be maintained by both the activities of spore vectors and differential competitive abilities on different hosts, but the influence of other competing microbes and micro-niche differentiation cannot be excluded.

Symbiotic yeasts from the mycangium, larval gut and woody substrate of an African stag beetle Xiphodontus antilope (Coleoptera: Lucanidae).

Female stag beetles (Lucanidae) possess internal mycangia to maintain microbial cultures. Yeasts from these mycangia may help with larval nutrition in nutrient poor woody substrates, but only a few Lucanidae taxa have been studied and all reports originate from Europe and Asia. We identify the first mycangial yeasts of a South African endemic Lucanidae beetle, Xiphodontus antilope, using nuclear ribosomal RNA and ITS DNA sequence data. In addition we identified yeasts from the larval gut, fecal matter, frass and woody substrate surrounding larvae and pupae. The mycangium of X. antilope was confined to females and is structurally similar to all other Lucanidae. Unlike most Lucanidae that seemingly associate with single species of yeast, or whose mycangia contain yeast monocultures, three yeast species were commonly isolated from X. antilope. Scheffersomyces coipomoensis was the most numerically dominant species on most substrates and in most individuals, but a second, undescribed, Scheffersomyces species was present in high numbers. A third species, also undescribed and unrelated to Scheffersomyces, was recovered from all mycangia but could not be detected in the larval gut, fecal matter, frass or woody substrates. We confirm a close association of Scheffersomyces yeasts with Lucanidae globally, but other taxa may also be involved. We show that the predominant mycangial yeasts also form the predominant yeasts within the larval gut and the woody substrates around the larvae and pupae. This combined external and internal colonization by the same yeasts may provide enhanced opportunities for nutrient acquisition, but this needs validation in future studies.

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.

Nitrogen-fixing bacteria and Oxalis - evidence for a vertically inherited bacterial symbiosis.

BACKGROUND: Plant-endophyte symbioses often revolve around nitrogen metabolism, and involve varying degrees of intimacy. Although evidence for vertical inheritance of nitrogen-fixing endophytic bacteria is increasing, it is confined mostly to crop plants, and to date no such system has been reported for geophytes. METHODS: Bacterial endophytes associated with Oxalis, the most species-rich geophytic genus form the Cape Flora in southern Africa was studied. Culturable endophytes were isolated from surface-sterilized vegetative and reproductive plant organs for six host species at three locations. Colonies of microbes on various artificial media were morphotyped, enumerated and identified using sequence data. Filter exclusion experiments were conducted to determine if endophytes were vertically transmitted to seeds, determine if mucilage plays a role to actively attract microbes from the soil and to assess microbial richness isolated from the mucilage of Oxalis seedlings. Fluorescent microscopy was implemented in order to visualize endophytic bacteria in cryo-sectioned seeds. RESULTS: Evidence for a novel, vertically transmitted symbiosis was reported. Communities of nitrogen-fixing and plant growth-promoting Bacillus endophytes were found to associate with selected Oxalis hosts from nitrogen-deficient environments of the Cape. Bacillus endophytes were ubiquitous and diverse across species and plant bodies, and were prominent in seeds. Three common nitrogen-fixing Bacillus have known oxalotrophic properties and appear to be housed inside specialised cavities (containing oxalates) within the plant body and seeds. CONCLUSIONS: The discovery of vertical transmission and potential benefits to both host and endophyte suggest a particularly tight mutualism in the Oxalis-endophyte system. This discovery suggests unexpected ways in which geophytes might avoid nitrogen deficiency, and suggest that such symbioses are more common than previously expected.

IMA Genome-F 11: Draft genome sequences of Fusarium xylarioides, Teratosphaeria gauchensis and T. zuluensis and genome annotation for Ceratocystis fimbriata.

Draft genomes of the fungal species Fusarium xylarioides, Teratosphaeria gauchensis and T. zuluensis are presented. In addition an annotation of the genome of Ceratocystis fimbriata is presented. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity and potential management strategies of these economically important fungi.

Teratosphaeria stem canker of Eucalyptus: two pathogens, one devastating disease.

BACKGROUND: Teratosphaeria gauchensis and T. zuluensis are closely related fungi that cause Teratosphaeria (previously Coniothyrium) stem canker disease on Eucalyptus species propagated in plantations for commercial purposes. This disease is present in many countries in which Eucalyptus trees are planted, and continues to spread with the international trade of infected plant germplasm. TAXONOMY: Fungi, Ascomycota, Pezizomycotina, Dothideomycetes, Dothideomycetidae, Capnodiales, Teratosphaeriaceae, Teratosphaeria. IDENTIFICATION: The causal agents form dark masses of pycnidia that are visible on the surface of distinct stem cankers that typically form on young green stem tissues. Accurate diagnosis of the causal agents requires DNA sequence data. HOST RANGE: Nine species of Eucalyptus are known to be affected. Of these, E. grandis and its hybrids, which include some of the most important planting stock globally, appear to be particularly vulnerable. DISEASE SYMPTOMS: Small necrotic lesions develop on young green stem tissue. These lesions coalesce to form large cankers that exude gum. Epicormic shoots develop below the girdling canker and, in severe cases, trees die. USEFUL WEBSITES: Mycobank, https://www.mycobank.org; Publications of the Forestry and Agricultural Biotechnology Institute (FABI), https://www.fabinet.up.ac.za/index.php/journals.

Genomic overview of closely related fungi with different Protea host ranges.

Genome comparisons of species with distinctive ecological traits can elucidate genetic divergence that influenced their differentiation. The interaction of a microorganism with its biotic environment is largely regulated by secreted compounds, and these can be predicted from genome sequences. In this study, we considered Knoxdaviesia capensis and Knoxdaviesia proteae, two closely related saprotrophic fungi found exclusively in Protea plants. We investigated their genome structure to compare their potential inter-specific interactions based on gene content. Their genomes displayed macrosynteny and were approximately 10 % repetitive. Both species had fewer secreted proteins than pathogens and other saprotrophs, reflecting their specialized habitat. The bulk of the predicted species-specific and secreted proteins coded for carbohydrate metabolism, with a slightly higher number of unique carbohydrate-degrading proteins in the broad host-range K. capensis. These fungi have few secondary metabolite gene clusters, suggesting minimal competition with other microbes and symbiosis with antibiotic-producing bacteria common in this niche. Secreted proteins associated with detoxification and iron sequestration likely enable these Knoxdaviesia species to tolerate antifungal compounds and compete for resources, facilitating their unusual dominance. This study confirms the genetic cohesion between Protea-associated Knoxdaviesia species and reveals aspects of their ecology that have likely evolved in response to their specialist niche.

Interactions among predators and plant specificity protect herbivores from top predators.

The worldwide loss of top predators from natural and agricultural systems has heightened the need to understand how important they are in controlling herbivore abundance. The effect of top predators on herbivore species is likely to depend on (1) the importance of the consumption of intermediate predators by top predators (intra-guild predation; IGP), but also on (2) plant specificity by herbivores, because specialists may defend themselves better (enemy-free space; EFS). Insectivorous birds, as top predators, are generally known to effectively control herbivorous insects, despite also consuming intermediate predators such as spiders, but how this effect varies among herbivore species in relation to the cascading effects of IGP and EFS is not known. To explore this, we excluded birds from natural fynbos vegetation in South Africa using large netted cages and recorded changes in abundance relative to control plots for 199 plant-dwelling intermediate predator and 341 herbivore morpho-species that varied in their estimated plant specificity. We found a strong negative effect of birds on the total abundance of all intermediate predators, with especially clear effects on spiders (strong IGP). In contrast with previous studies, which document a negative effect of birds on herbivores, we found an overall neutral effect of birds on herbivore abundance, but the effect varied among species: some species were negatively affected by birds, suggesting that they were mainly consumed by birds, whereas others, likely released from spiders by IGP, were positively affected. Some species were also effectively neutrally affected by birds. These tended to be more specialized to plants compared to the other species, which may imply that some plant specialists benefited from protection provided by EFS from both birds and spiders. These results suggest that the response of herbivore species to top predators may depend on cascading effects of interactions among predators and on their degree of plant specificity.

Biodiversity and ecology of flower-associated actinomycetes in different flowering stages of Protea repens.

Actinomycete bacteria have previously been reported from reproductive structures (infructescences) of Protea (sugarbush/suikerbos) species, a niche dominated by fungi in the genera Knoxdaviesia and Sporothrix. It is probable that these taxa have symbiotic interactions, but a lack of knowledge regarding their diversity and general ecology precludes their study. We determined the diversity of actinomycetes within Protea repens inflorescence buds, open inflorescences, young and mature infructescences, and leaf litter surrounding these trees. Since the P. repens habitat is fire-prone, we also considered the potential of these bacteria to recolonise infructescences after fire. Actinomycetes were largely absent from flower buds and inflorescences but were consistently present in young and mature infructescences. Two Streptomyces spp. were the most consistent taxa recovered, one of which was also routinely isolated from leaf litter. Lower colonisation rates were evident in samples from a recently burnt site. One of the most consistent taxa isolated from older trees in the unburnt site was absent from this site. Our findings show that P. repens has a distinct community of actinomycetes dominated by a few species. These communities change over time and infructescence developmental stage, season and the age of the host population. Mature infructescences appear to be important sources of inoculum for some of the actinomycetes, seemingly disrupted by fire. Increased fire frequency limiting maturation of P. repens infructescences could thus impact future actinomycete colonisation in the landscape. Streptomyces spp. are likely to share this niche with the ophiostomatoid fungi, which merits further study regarding their interactions and mode of transfer.

Two new Sporothrix species from Protea flower heads in South African Grassland and Savanna.

The inflorescences and infructescences of African Protea trees provide habitat for a large diversity of Sporothrix species. Here we describe two additional members, Sporothrix nsini sp. nov. and Sporothrix smangaliso sp. nov., that are associated with the infructescences of various Protea species from grasslands and savannas in the KwaZulu-Natal, North-West, Gauteng and Mpumalanga provinces of South Africa. Their description raises the number of described Protea-associated Sporothrix species to twelve. S. smangaliso sp. nov. is distantly related to other Protea-associated species and, in phylogenies using multiple markers (ITS, beta-tubulin and calmodulin), groups with taxa such as Sporothrix bragantina from Brazil and Sporothrix curviconia from the Ivory Coast. S. nsini sp. nov. resolved as sister to a clade containing four other Protea-associated species within the Sporothrix stenoceras complex. S. nsini sp. nov. was collected from within the same infructescences of Protea caffra that also contained the closely related S. africana and S. protearum. This highlights the need to study and understand the factors that influence host selection and speciation of Sporothrix in this atypical niche.

Birds Mediate a Fungus-Mite Mutualism.

Mutualisms between ophiostomatoid fungi and arthropods have been well documented. These fungi commonly aid arthropod nutrition and, in turn, are transported to new niches by these arthropods. The inflorescences of Protea trees provide a niche for a unique assemblage of ophiostomatoid fungi. Here, mites feed on Sporothrix fungi and vector the spores to new niches. Protea-pollinating beetles transport the spore-carrying mites between Protea trees. However, many Protea species are primarily pollinated by birds that potentially play a central role in the Protea-Sporothrix-mite system. To investigate the role of birds in the movement of mites and/or fungal spores, mites were collected from Protea inflorescences and cape sugarbirds, screened for Sporothrix fungal spores and tested for their ability to feed and reproduce on the fungal associates. Two mite species where abundant in both Protea inflorescences and on cape sugarbirds and regularly carried Sporothrix fungal spores. One of these mite species readily fed and reproduced on its transported fungal partner. For dispersal, this mite (a Glycyphagus sp.) attached to a larger mite species (Proctolaelaps vandenbergi) which, in turn, were carried by the birds to new inflorescences. The results of this study provide compelling evidence for a new mite-fungus mutualism, new mite-mite commensalisms and the first evidence of birds transporting mites with Sporothrix fungal spores to colonise new Protea trees.