Fungal radiation in the Cape Floristic Region: an analysis based on Gondwanamyces and Ophiostoma

The Cape Floristic Region (CFR) displays high levels of plant diversity and endemism, and has received focused botanical systematic attention. In contrast, fungal diversity patterns and co-evolutionary processes in this region have barely been investigated. Here we reconstruct molecular phylogenies using the ITS and beta-tubulin gene regions of the ophiostomatoid fungi Gondwanamyces and Ophiostoma associated with southern African Protea species. Results indicate that they evolved in close association with Protea. In contrast to Protea, Ophiostoma species migrated to the CFR from tropical and subtropical Africa, where they underwent subsequent radiation. In both Gondwanamyces and Ophiostoma vector arthropods probably facilitated long-distance migration and shorter-distance dispersal. Although ecological parameters shaped most associations between ophiostomatoid fungi and Protea, there is congruence between fungal-host-associations and the systematic classification of Protea. These results confirm that the entire biotic environment must be considered in order to understand diversity and evolution in the CFR as a whole.     

Ophiostoma gemellus and Sporothrix variecibatus from mites infesting Protea infructescences in South Africa

Ophiostoma (Ophiostomatales) represents a large genus of fungi mainly known from associations with bark beetles (Curculionidae: Scolytinae) infesting conifers in the northern hemisphere. Few southern hemisphere native species are known, and the five species that consistently occur in the infructescences of Protea spp. in South Africa are ecologically unusual. Little is known about the vectors of Ophiostoma spp. from Protea infructescences, however recent studies have considered the possible role of insects and mites in the distribution of these exceptional fungi. In this study we describe a new species of Ophiostoma and a new Sporothrix spp. with affinities to Ophiostoma, both initially isolated from mites associated with Protea spp. They are described as Ophiostoma gemellus sp. nov. and Sporothrix variecibatus sp. nov. based on their morphology and comparisons of DNA sequence data of the 28S ribosomal, beta-tubulin and internal transcribed spacer (ITS1, 5.8S, ITS2) regions. DNA sequences of S. variecibatus were identical to those of a Sporothrix isolate obtained from Eucalyptus leaf litter in the same area in which S. variecibatus occurs in Protea infructescences. Results of this study add evidence to the view that mites are the vectors of Ophiostoma spp. that colonize Protea infructescences. They also show that DNA sequence comparisons are likely to reveal additional cryptic species of Ophiostoma in this unusual niche.     

Mites are the most common vectors of the fungus Gondwanamyces proteae in Protea infructescences

Entomochoric spore dispersal is well-documented for most ophiostomatoid fungal genera, most of which are associated with bark or ambrosia beetles. Gondwanamyces spp. are unusual members of this group that were first discovered in the flower heads of the primitive angiosperm genus Protea, that is mostly restricted to the Cape Floristic region of Africa. In this study, we present the discovery of the vectors of Gondwanamyces proteae in Protea repens infructescences, which were identified using PCR, direct isolation, and light microscopy. Gondwanamyces proteae DNA and ascospores were identified on diverse lineages of arthropods including beetles (Euderes lineicolis and Genuchus hottentottus), bugs (Oxycarenus maculates), a psocopteran species and five mite (Acari) species. Based on isolation frequency, however, a mite species in the genus Trichouropoda appears to be the most common vector of G. proteae. Gondwanamyces spores were frequently observed within pit mycangia at the base of the legs of these mites. Manipulative experiments demonstrated the ability of mites to carry viable G. proteae spores whilst in transit on the beetle G. hottentottus and that these mites are able to transfer G. proteae spores to uncolonised substrates in vitro. Interestingly, this same mite species has also been implicated as vector of Ophiostoma spores on P. repens and belongs to the same genus of mites that vector Ophiostoma spp. associated with pine-infesting bark beetles in the Northern Hemisphere.     

Discovery of fungus-mite mutualism in a unique niche

The floral heads (infructescences) of South African Protea L. represent a most unusual niche for fungi of the economically important genus Ophiostoma Syd. and P. Syd. emend. Z.W. de Beer et al. Current consensus holds that most members of Ophiostoma are vectored by tree-infesting bark beetles. However, it has recently been suggested that mites, phoretic on these bark beetles, may play a central role in the dispersal of Ophiostoma. No bark beetles are known from Protea. Therefore, identifying the vectors of Ophiostoma in Protea infructescences would independently evaluate the role of various arthropods in the dispersal of Ophiostoma. Infructescence-colonizing arthropods were tested for the presence of Ophiostoma DNA using polymerase chain reaction (PCR) and for reproductive propagules by isolation on agar plates. PCR tests revealed that few insects carried Ophiostoma DNA. In contrast, various mites (Proctolaelaps vandenbergi Ryke, two species of Tarsonemus Canestrini and Fonzago, and one Trichouropoda Berlese species) frequently carried Ophiostoma propagules. DNA sequence comparisons for 28S ribosomal DNA confirmed the presence of O. splendens G. J. Marais and M. J. Wingf., O. palmiculminatum Roets et al., and O. phasma Roets et al. on these mites. Two apparently undescribed species of Ophiostoma were also identified. Light and scanning electron microscopy revealed specialized structures in Trichouropoda and one Tarsonemus sp. that frequently contained Ophiostoma spores. The Trichouropoda sp. was able to complete its life cycle on a diet consisting solely of its identified phoretic Ophiostoma spp. This study provides compelling evidence that mites are the primary vectors of infructescence-associated Ophiostoma spp. in South Africa.     

New species of Gondwanamyces from dying Euphorbia trees in South Africa

Gondwanamyces and its Custingophora anamorphs were first described from Protea infructescences in South Africa. Subsequently these unusual fungi were also found on Cecropia in Central America. During an investigation into the decline and death of native Euphorbia trees in South Africa, several fungal isolates resembling the anamorph state of Gondwanamyces were obtained from diseased tissues. In this study these isolates are identified based on morphology and comparisons of DNA sequences. Two previously unknown Gondwanamyces species were identified, both were associated with damage caused by beetles (Cossonus sp.). Inoculation studies showed that the new species of Gondwanamyces are pathogenic on Euphorbia ingens and may contribute to the decline of these trees.     

First fungal community analyses of endophytic ascomycetes associated with Viscum album ssp. austriacum and its host Pinus sylvestris

The endophytic fungal communities in the hemi-parasitic epiphyte Viscum album and in its phorophyte Pinus sylvestris were compared to reveal the fungal distribution patterns in their hosts. The ITS nrDNA of 208 multiple-isolated fungal strains was sequenced and a newly designed process was applied for assigning taxon names to the obtained sequences. Furthermore, the isolates were grouped as clusters, by subjecting a sequence similarity matrix to various cluster analyses, the results of which were compared and verified by data from phylogenetic reconstructions. In contrast to a previously reported dominance of Leotiomycetes among Pinus inhabiting fungi, the endophytic communities of the two host plant species studied here were dominated by Xylariaceae (Sordariomycetes). This is in accordance with the finding that host selectivity was only a minor factor in explaining the distribution patterns of the endophytic fungi in Viscum and Pinus. Organ and, probably, tissue selectivity had a more pronounced effect. The composition and condition of the woods in the surrounding, however, are concluded to be the major determinants, due to the following circumstantial evidence: The highest similarities in fungal community compositions were found for the leaves of the two host plant species, especially when considering only the older leaves. The finding that the inhabitants of matured or senescent organs are less host-selective is in accordance with decreasing defence capabilities of ageing host plant tissue and an increased nutrient supply for saprobic taxa. Therefore, the composition of the fungal communities in ageing leaves seems to be predominantly ascribed to contagious spread and to depend on the spectrum of nearby sporulating fungal taxa. We suggest that because a broad range of suitable substrates for Xylariaceae was present in immediate vicinity of the study sites, these fungi also dominated among the recorded endophytic taxa.     

Diversity of saprobic microfungi

The data needed to derive an accurate estimate of saprobic microfungi are insufficient, incomplete and contradictory. We therefore address issues that will ultimately reveal whether there are 1.5 million global fungal species, which is the generally accepted working estimate. Our data indicates that large numbers of fungi occur on host families, such as Musaceae, host genera such as Nothofagus and individual host species such as Eucalyptus globulus, and that fungi may be specific or recurrent on different plant groups. Recent studies have shown that fungal numbers on hosts may be larger than originally thought as saprobes are organ-specific/-recurrent and changes in fungal communities occur as substrata decays. Other issues, such as the impact of geography, of methodology and of taxonomy are also addressed. There is evidence that fungi on the same host at different locations also differs; site-specific factors and geographic distance may be more important than host/substrate in shaping fungal assemblages. Methodology impacts on estimates of species diversity with many more taxa observed using indirect isolation protocols as compared to direct isolations from leaves. Our understanding of fungal species numbers in speciose genera is important. In some fungal groups accepted species have been reduced to a few species, while in other groups many cryptic species are being uncovered. While we make a number of generalisations from the studies reported here, this review also highlights some of the limitations mycologists currently have to contend with. A large body of knowledge exists for certain groups of microfungi or for microfungi occurring on certain substrata/hosts. However, it is likely that we are drawing conclusions from data that are somewhat biased toward fungi and host/substrata that are of interest to human endeavours. The discrepancy between the numbers of fungi described from only one economically important genus, Eucalyptus, and all the other members of the Myrtaceae is but one example of this bias. By incorporating the large body of work that is already available and adding appropriate complementary studies, we can accelerate our understanding of microfungal diversity and this will eventually lead us to a realistic estimate of global fungal species numbers.     

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.     

Understanding context-dependency in plant-microbe symbiosis: The influence of abiotic and biotic contexts on host fitness and the rate of symbiont transmission

Understanding the dynamics of a hereditary symbiosis requires testing how ecological factors alter not only the fitness consequences of the symbiosis, but also the rate of symbiont transmission to the next generation. The relative importance of these two mechanisms remains unresolved because studies have not simultaneously examined how the ecological context of the symbiosis influences both costs/benefits and the rate of vertical transmission. Fungal endophytes in grasses have provided particularly tractable systems for investigating the ecological and evolutionary dynamics of hereditary symbiosis. Here we examine interactions between a fungal endophyte, Epichloë amarillans, and its grass host, Agrostis hyemalis, under altered abiotic and biotic contexts: a gradient of water availability and in the presence versus absence of soil microbes. We show that benefits of the symbiosis were strongest when water was limiting. Symbiotic plants at the lowest watering level produced ∼40% more inflorescences and greater seed mass than non-symbiotic plants, while at the highest watering level, symbiotic and non-symbiotic plants did not significantly differ in reproductive fitness. Benefits appear to accrue by allowing hosts to escape from drought, a response that has not been previously reported to be endophyte-mediated. Symbiotic plants at the lowest watering level flowered 9 days earlier than non-symbiotic plants. Interestingly, our results suggest the symbiosis may be costly in the presence of soil microbes, as on live soil, the biomass of symbiotic plants was lower than the biomass of symbiont-free plants. We detected no effect of either the biotic or abiotic context on the rate of symbiont vertical transmission, suggesting that the context-dependent benefits of the symbiosis are the more important driver of variation in symbiont frequency in this system.     

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.     

Basidiobolus haptosporus is frequently associated with the gamasid mite Leptogamasus obesus

Two species of mites inhabiting a pine forest soil were screened for associated fungi. The fungal community composition was assessed in 49 mite and 19 soil samples by environmental PCR with a focus on fungi of the genus Basidiobolus. PCR products of the fungal ITS rRNA gene were analyzed by sub-cloning, RFLP-analysis, and sequencing. Thereby Basidiobolus haptosporus was found for the first time to be frequently associated with the gamasid mite species Leptogamasus obesus, while being absent from the oribatid mite Oppiella subpectinata, and from the surrounding soil. The fungus was isolated in pure culture for a detailed morphological characterization and experimental approaches concerning the nature of this fungus-mite association. The experiments and a supporting microscopic screening of freshly captured gamasid mites revealed no indications for the fungus being localized in the mites' gut or haemocoel, but a single spore was found attached to an individual of L. obesus. However, an exclusive phoretic association does not satisfactorily explain the frequent detection of B. haptosporus DNA on or in L. obesus, and the absence of the fungus from soil samples seems not to be in line with its assumed ecology as a widespread saprobic soil fungus. Therefore, a second host species in the life cycle of B. haptosporus is discussed as a working hypothesis.     

Botryosphaeriaceae species overlap on four unrelated,native South African hosts

Botryosphaeriaceae represents an important and diverse family of latent fungal pathogens of woody plants. We address the question of host range of these fungi by sampling leaves and branches of four native South African trees, including Acacia karroo (Fabaceae), Celtis africana (Cannabaceae), Searsia lancea (Anacardiaceae), and Gymnosporia buxifolia (Celastraceae). Two new species of the Botryosphaeriaceae, namely Tiarosporella africana sp. nov. and Aplosporella javeedii sp. nov. were identified, together with five known species, including Neofusicoccum parvum, Neofusicoccum kwambonambiense, Spencermartinsia viticola, Diplodia pseudoseriata, and Botryosphaeria dothidea. Most Botryosphaeriaceae occurred on more than one host. With the exception of S. lancea, which was infected by A. javeedii all the hosts were infected by more than one Botryosphaeriaceae species. Collectively, the results suggest that some intrinsic host factors, possibly combined with local environmental conditions, affect the distribution and co-infectivity of various hosts by the Botryosphaeriaceae. This would counteract the general ability of a species in the Botryosphaeriaceae to infect a broad range of plants. The combination of host and environmental factors might also explain why some Botryosphaeriaceae with apparently broad host ranges, are found on different suites of hosts in different areas of the world.     

Mould growth on building materials under low water activities. Influence of humidity and temperature on fungal growth and secondary metabolism

The influence of relative humidity (RH) and temperature on growth and metabolism of eight microfungi on 21 different types of building material was investigated. The fungi were applied as a dry mixture to the materials, which were incubated at 5°C, 10°C, 20°C and 25°C at three humidity levels in the range 69–95% RH over 4–7 months. The lower limit for fungal growth on wood, wood composites and starch-containing materials was 78% RH at 20–25°C and increased to 90% RH at 5°C. An RH of 86% was necessary for growth on gypsum board. Ceramic materials supported growth at RH >90%, although 95% RH was needed to yield chemically detectable quantities of biomass. Almost exclusively only Penicillium, Aspergillus and Eurotium (contaminant) species grew on the materials. Production of secondary metabolites and mycotoxins decreased with humidity and the quantities of metabolites were insignificant compared with those produced at high RH (RH >95%), except in the case of Eurotium.     

Biodiversity and ecology of flower-associated actinomycetes in different flowering stages of <Emphasis Type="Italic">Protea repens</Emphasis>

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.     

Developing Aspergillus as a host for heterologous expression

Filamentous fungi have long been used for production of a range of valuable products; with the advent of molecular biology, it became apparent that these fungi possess considerable potential as expression hosts for the production of heterologous proteins and small molecules. Aspergillus is an important genus, including well known species of economically significant molds, and widely used for basic genetic research. The development of a genetic engineering "toolkit" for Aspergillus, such as those existing for the simpler yeasts and bacteria, was delayed due to the added complexity of the filamentous fungi, and also to the lesser resources devoted to their study. History of the development of Aspergillus as an expression host, current state of the art and future directions are reviewed, touching on related research in other fungi when discussing the areas of greatest potential for future biotechnological applications, focusing on the large and diverse families of fungal secondary metabolites.     

Evolution of entomopathogenicity in fungi

The recent completions of publications presenting the results of a comprehensive study on the fungal phylogeny and a new classification reflecting that phylogeny form a new basis to examine questions about the origins and evolutionary implications of such major habits among fungi as the use of living arthropods or other invertebrates as the main source of nutrients. Because entomopathogenicity appears to have arisen or, indeed, have lost multiple times in many independent lines of fungal evolution, some of the factors that might either define or enable entomopathogenicity are examined. The constant proximity of populations of potential new hosts seem to have been a factor encouraging the acquisition or loss of entomopathogenicity by a very diverse range of fungi, particularly when involving gregarious and immobile host populations of scales, aphids, and cicadas (all in Hemiptera). An underlying theme within the vast complex of pathogenic and parasitic ascomycetes in the Clavicipitaceae (Hypocreales) affecting plants and insects seems to be for interkingdom host-jumping by these fungi from plants to arthropods and then back to the plant or on to fungal hosts. Some genera of Entomophthorales suggest that the associations between fungal pathogens and their insect hosts appear to be shifting away from pathogenicity and towards nonlethal parasitism.     

Host and geographic structure of endophytic and endolichenic fungi at a continental scale

? Premise of the study: Endophytic and endolichenic fungi occur in healthy tissues of plants and lichens, respectively, playing potentially important roles in the ecology and evolution of their hosts. However, previous sampling has not comprehensively evaluated the biotic, biogeographic, and abiotic factors that structure their communities. ? Methods: Using molecular data we examined the diversity, composition, and distributions of 4154 endophytic and endolichenic Ascomycota cultured from replicate surveys of ca. 20 plant and lichen species in each of five North American sites (Madrean coniferous forest, Arizona; montane semideciduous forest, North Carolina; scrub forest, Florida; Beringian tundra and forest, western Alaska; subalpine tundra, eastern central Alaska). ? Key results: Endolichenic fungi were more abundant and diverse per host species than endophytes, but communities of endophytes were more diverse overall, reflecting high diversity in mosses and lycophytes. Endophytes of vascular plants were largely distinct from fungal communities that inhabit mosses and lichens. Fungi from closely related hosts from different regions were similar in higher taxonomy, but differed at shallow taxonomic levels. These differences reflected climate factors more strongly than geographic distance alone. ? Conclusions: Our study provides a first evaluation of endophytic and endolichenic fungal associations with their hosts at a continental scale. Both plants and lichens harbor abundant and diverse fungal communities whose incidence, diversity, and composition reflect the interplay of climatic patterns, geographic separation, host type, and host lineage. Although culture-free methods will inform future work, our study sets the stage for empirical assessments of ecological specificity, metabolic capability, and comparative genomics.     

Geographic structure in a widespread plant-mycorrhizal interaction: pines and false truffles

Mutualistic interactions are likely to exhibit a strong geographic mosaic in their coevolutionary dynamics, but the structure of geographic variation in these interactions is much more poorly characterized than in host-parasite interactions. We used a cross-inoculation experiment to characterize the scales and patterns at which geographic structure has evolved in an interaction between three pine species and one ectomycorrhizal fungus species along the west coast of North America. We found substantial and contrasting patterns of geographic interaction structure for the plants and fungi. The fungi exhibited a clinal pattern of local adaptation to their host plants across the geographic range of three coastal pines. In contrast, plant growth parameters were unaffected by fungal variation, but varied among plant populations and species. Both plant and fungal performance measures varied strongly with latitude. This set of results indicates that in such widespread species interactions, interacting species may evolve asymmetrically in a geographic mosaic because of differing evolutionary responses to clinally varying biotic and abiotic factors.     

Trade-offs between arbuscular mycorrhizal fungal competitive ability and host growth promotion in Plantago lanceolata

In this study we tested for trade-offs between the benefit arbuscular mycorrhizal (AM) fungi provide for hosts and their competitive ability in host roots, and whether this potential trade-off shifts in the presence of a plant stress (herbivory). We used three species of AM fungi previously determined to vary in host growth promotion and spore production in association with host plants. We found that these AM fungal species competed for root space, and the best competitor, Scutellospora calospora, was the worst mutualist. In addition, the worst competitor, Glomus white, was the best mutualist. Competition proved to have stronger effects on fungal infection patterns than herbivory, and competitive dominance was not altered by herbivory. We found a similar pattern in a previous test of competition among AM fungi, and we discuss the implications of these results for the persistence of the mutualism and feedbacks between AM fungi and their plant hosts.