Sources of Fungal Symbionts in the Microbiome of a Mobile Insect Host, Spodoptera frugiperda.

The sources of fungal symbionts of insects are not well understood, yet the acquisition and assembly of fungal communities in mobile insect hosts have important implications for the ecology of migratory insects and their plant hosts. To determine potential sources of fungi associated with the fall armyworm (Spodoptera frugiperda), we characterized the fungal communities associated with four different ecological compartments (insects, infested leaves, uninfested leaves, and soil) and estimated the contributions of each of these potential sources to the insect's fungal microbiome. Results show that insect fungal community composition was distinct from and more varied than the composition of fungal communities in the environment of those insects (plants and soil). Among the sources evaluated, on average we found a surprisingly large apparent contribution from other congeneric S. frugiperda insect larvae (ca. 25%) compared to the contribution from soil or plant sources (< 5%). However, a large proportion of the insect microbiome could not be attributed to the sampled sources and was instead attributed to unknown sources (ca. 50%). Surprisingly, we found little evidence for exchange of fungal taxa, with the exception of a Fusarium oxysporum and a Cladosporium sp. OTU, between larvae and the infested leaves on which they fed. Together, our results suggest that mobile insects such as S. frugiperda obtain their fungal symbionts from a variety of sources, not limited to plants and soil, but including conspecific insects and other unsampled environmental sources, and that transmission among insects may play an important role in acquisition of fungal symbionts.

Do Interactions among Microbial Symbionts Cause Selection for Greater Pathogen Virulence?

AbstractThe ecological and evolutionary consequences of microbiome treatments aimed at protecting plants and animals against infectious disease are not well understood, even as such biological control measures become more common in agriculture and medicine. Notably, we lack information on the impacts of symbionts on pathogen fitness with which to project the consequences of competition for the evolution of virulence. To address this gap, we estimated fitness consequences for a common plant pathogen, Ustilago maydis, over differing virulence levels and when the host plant (Zea mays) is coinfected with a defensive symbiont (Fusarium verticillioides) and compared these fitness estimates to those obtained when the symbiont is absent. Here, virulence is measured as the reduction in the growth of the host caused by pathogen infection. Results of aster statistical models demonstrate that the defensive symbiont most negatively affects pathogen infection and that these effects propagate through subsequent stages of disease development to cause lower pathogen fitness across all virulence levels. Moreover, the virulence level at which pathogen fitness is maximal is higher in the presence of the defensive symbiont than in its absence. Thus, as expected from theory for multiple parasites, competition from the defensive symbiont may cause selection for increased pathogen virulence. More broadly, we consider that the evolutionary impacts of interactions between pathogens and microbial symbionts will depend critically on biological context and environment and that interactions among diverse microbial symbionts in spatially heterogeneous communities contribute to the maintenance of the highly diverse symbiotic functions observed in these communities.

Defensive Symbiosis and the Evolution of Virulence.

AbstractA microbiome rife with enemies of the host should cause selection for defensive traits in symbionts, yet such complex environments are also predicted to select for greater symbiont virulence. Why then do we so often observe defensive mutualists that protect hosts while causing little to no damage? To address this question, we build a symbiont-centered model that incorporates the evolution of two independent symbiont traits: defense and virulence. Virulence is modeled as a continuous trait spanning parasitism (positive virulence) and mutualism (negative virulence), thus accounting for the entire range of direct effects that symbionts have on host mortality. Defense is modeled as a continuous trait that ameliorates the costs to the host associated with infection by a deleterious parasite. We show that the evolution of increased defense in one symbiont may lead to the evolution of lower virulence in both symbionts and even facilitate pathogens evolving to mutualism. However, results are context dependent, and when defensive traits are costly, the evolution of greater defense may also lead to the evolution of greater virulence, breaking the common expectation that defensive symbionts are necessarily mutualists toward the host.

T-BAS: Tree-Based Alignment Selector toolkit for phylogenetic-based placement, alignment downloads and metadata visualization: an example with the Pezizomycotina tree of life.

Motivation: High-quality phylogenetic placement of sequence data has the potential to greatly accelerate studies of the diversity, systematics, ecology and functional biology of diverse groups. We developed the Tree-Based Alignment Selector (T-BAS) toolkit to allow evolutionary placement and visualization of diverse DNA sequences representing unknown taxa within a robust phylogenetic context, and to permit the downloading of highly curated, single- and multi-locus alignments for specific clades. Results: In its initial form, T-BAS v1.0 uses a core phylogeny of 979 taxa (including 23 outgroup taxa, as well as 61 orders, 175 families and 496 genera) representing all 13 classes of largest subphylum of Fungi-Pezizomycotina (Ascomycota)-based on sequence alignments for six loci (nr5.8S, nrLSU, nrSSU, mtSSU, RPB1, RPB2 ). T-BAS v1.0 has three main uses: (i) Users may download alignments and voucher tables for members of the Pezizomycotina directly from the reference tree, facilitating systematics studies of focal clades. (ii) Users may upload sequence files with reads representing unknown taxa and place these on the phylogeny using either BLAST or phylogeny-based approaches, and then use the displayed tree to select reference taxa to include when downloading alignments. The placement of unknowns can be performed for large numbers of Sanger sequences obtained from fungal cultures and for alignable, short reads of environmental amplicons. (iii) User-customizable metadata can be visualized on the tree. Availability and Implementation: T-BAS Version 1.0 is available online at http://tbas.hpc.ncsu.edu . Registration is required to access the CIPRES Science Gateway and NSF XSEDE's large computational resources. Contact: icarbon@ncsu.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Coevolution between Mutualists and Parasites in Symbiotic Communities May Lead to the Evolution of Lower Virulence.

Most eukaryotes harbor a diverse community of parasitic, mutualistic, and commensal microbial symbionts. Although the diversity of these microbial symbiotic communities has recently drawn considerable attention, theory regarding the evolution of interactions among symbionts and with the host is still in its nascent stages. Here we evaluate the role of interactions among coinfecting symbionts in the evolution of symbiont virulence toward the host. To do so, we place the virulence-transmission trade-off into a community context and model the evolution of symbiont trophic modes along the continuum from parasitism (virulence) to mutualism (negative virulence). We establish a framework for studying multiple infections of a host by the same symbiont species and coinfection by multiple species, using a concept of shared costs, wherein the negative consequences of virulence (or harm) toward the host are shared among symbionts. Our results show that mutualism can be maintained under infection by multiple symbionts when shared costs are sufficiently low, while greater virulence and parasitism toward the host are more likely when shared costs are high. Last, for coinfection by more than one species, we show that if the presence of a mutualist ameliorates some of the costs of pathogen virulence, then the symbiotic community may more often evolve to a more commensal state and maintain mutualisms.

Disentangling environmental and host sources of fungal endophyte communities in an experimental beachgrass study.

Disentangling the ecological factors that contribute to the assembly of the microbial symbiont communities within eukaryotic hosts is an ongoing challenge. Broadly speaking, symbiont propagules arrive either from external sources in the environment or from internal sources within the same host individual. To understand the relative importance of these propagule sources to symbiont community assembly, we characterized symbiotic fungal endophyte communities within the roots of three species of beachgrass in a field experiment. We manipulated two aspects of the external environment, successional habitat and physical disturbance. To determine the role of internal sources of propagules for endophyte community assembly, we used beachgrass individuals with different pre-existing endophyte communities. Endophyte species richness and community composition were characterized using culture-based and next-generation sequencing approaches. Our results showed that external propagule sources associated with successional habitat, but not disturbance, were particularly important for colonization of most endophytic taxa. In contrast, internal propagule sources played a minor role for most endophytic taxa but were important for colonization by the dominant taxon Microdochium bolleyi. Our findings highlight the power of manipulative field experiments to link symbiont community assembly to its underlying ecological processes, and to ultimately improve predictions of symbiont community assembly across environments.

Plant Host Species and Geographic Distance Affect the Structure of Aboveground Fungal Symbiont Communities, and Environmental Filtering Affects Belowground Communities in a Coastal Dune Ecosystem.

Microbial symbionts inhabit tissues of all plants and animals. Their community composition depends largely on two ecological processes: (1) filtering by abiotic conditions and host species determining the environments that symbionts are able to colonize and (2) dispersal-limitation determining the pool of symbionts available to colonize a given host and community spatial structure. In plants, the above- and belowground tissues represent such distinct habitats for symbionts that we expect different effects of filtering and spatial structuring on their symbiont communities. In this study, we characterized above- and belowground communities of fungal endophytes--fungi living asymptomatically within plants--to understand the contributions of filtering and spatial structure to endophyte community composition. We used a culture-based approach to characterize endophytes growing in leaves and roots of three species of coastal beachgrasses in dunes of the USA Pacific Northwest. For leaves, endophyte isolation frequency and OTU richness depended primarily on plant host species. In comparison, for roots, both isolation frequency and OTU richness increased from the nutrient-poor front of the dune to the higher-nutrient backdune. Endophyte community composition in leaves exhibited a distance-decay relationship across the region. In a laboratory assay, faster growth rates and lower spore production were more often associated with leaf- than root-inhabiting endophytes. Overall, our results reveal a greater importance of biotic filtering by host species and dispersal-limitation over regional geographic distances for aboveground leaf endophyte communities and stronger effects of abiotic environmental filtering and locally patchy distributions for belowground root endophyte communities.

Genetic variation in horizontally transmitted fungal endophytes of pine needles reveals population structure in cryptic species.

UNLABELLED: • PREMISE OF THE STUDY: Fungal endophytes comprise one of the most ubiquitous groups of plant symbionts, inhabiting healthy leaves and stems of all major lineages of plants. Together, they comprise immense species richness, but little is known about the fundamental processes that generate their diversity. Exploration of their population structure is needed, especially with regard to geographic distributions and host affiliations.• METHODS: We take a multilocus approach to examine genetic variation within and among populations of Lophodermium australe, an endophytic fungus commonly associated with healthy foliage of pines in the southeastern United States. Sampling focused on two pine species ranging from montane to coastal regions of North Carolina and Virginia.• KEY RESULTS: Our sampling revealed two genetically distinct groups within Lophodermium australe. Our analysis detected less than one migrant per generation between them, indicating that they are distinct species. The species comprising the majority of isolates (major species) demonstrated a panmictic structure, whereas the species comprising the minority of isolates (cryptic species) demonstrated isolation by distance. Distantly related pine species hosted the same Lophodermium species, and host species did not influence genetic structure.• CONCLUSIONS: We present the first evidence for isolation by distance in a foliar fungal endophyte that is horizontally transmitted. Cryptic species may be common among microbial symbionts and are important to delimit when exploring their genetic structure and microevolutionary processes. The hyperdiversity of endophytic fungi may be explained in part by cryptic species without apparent ecological and morphological differences as well as genetic diversification within rare fungal species across large spatial scales.

Environmental drivers and cryptic biodiversity hotspots define endophytes in Earth's largest terrestrial biome.

Understanding how symbiotic associations differ across environmental gradients is key to predicting the fate of symbioses as environments change, and it is vital for detecting global reservoirs of symbiont biodiversity in a changing world.1,2,3 However, sampling of symbiotic partners at the full-biome scale is difficult and rare. As Earth's largest terrestrial biome, boreal forests influence carbon dynamics and climate regulation at a planetary scale. Plants and lichens in this biome host the highest known phylogenetic diversity of fungal endophytes, which occur within healthy photosynthetic tissues and can influence hosts' resilience to stress.4,5 We examined how communities of endophytes are structured across the climate gradient of the boreal biome, focusing on the dominant plant and lichen species occurring across the entire south-to-north span of the boreal zone in eastern North America. Although often invoked for understanding the distribution of biodiversity, neither a latitudinal gradient nor mid-domain effect5,6,7 can explain variation in endophyte diversity at this trans-biome scale. Instead, analyses considering shifts in forest characteristics, Picea biomass and age, and nutrients in host tissues from 46° to 58° N reveal strong and distinctive signatures of climate in defining endophyte assemblages in each host lineage. Host breadth of endophytes varies with climate factors, and biodiversity hotspots can be identified at plant-community transitions across the boreal zone at a global scale. Placed against a backdrop of global circumboreal sampling,4 our study reveals the sensitivity of endophytic fungi, their reservoirs of biodiversity, and their important symbiotic associations, to climate.

Cryptic functional diversity within a grass mycobiome.

Eukaryotic hosts harbor tremendously diverse microbiomes that affect host fitness and response to environmental challenges. Fungal endophytes are prominent members of plant microbiomes, but we lack information on the diversity in functional traits affecting their interactions with their host and environment. We used two culturing approaches to isolate fungal endophytes associated with the widespread, dominant prairie grass Andropogon gerardii and characterized their taxonomic diversity using rDNA barcode sequencing. A randomly chosen subset of fungi representing the diversity of each leaf was then evaluated for their use of different carbon compound resources and growth on those resources. Applying community phylogenetic analyses, we discovered that these fungal endophyte communities are comprised of phylogenetically distinct assemblages of slow- and fast-growing fungi that differ in their use and growth on differing carbon substrates. Our results demonstrate previously undescribed and cryptic functional diversity in carbon resource use and growth in fungal endophyte communities of A. gerardii.

Globally consistent response of plant microbiome diversity across hosts and continents to soil nutrients and herbivores.

All multicellular organisms host a diverse microbiome composed of microbial pathogens, mutualists, and commensals, and changes in microbiome diversity or composition can alter host fitness and function. Nonetheless, we lack a general understanding of the drivers of microbiome diversity, in part because it is regulated by concurrent processes spanning scales from global to local. Global-scale environmental gradients can determine variation in microbiome diversity among sites, however an individual host's microbiome also may reflect its local micro-environment. We fill this knowledge gap by experimentally manipulating two potential mediators of plant microbiome diversity (soil nutrient supply and herbivore density) at 23 grassland sites spanning global-scale gradients in soil nutrients, climate, and plant biomass. Here we show that leaf-scale microbiome diversity in unmanipulated plots depended on the total microbiome diversity at each site, which was highest at sites with high soil nutrients and plant biomass. We also found that experimentally adding soil nutrients and excluding herbivores produced concordant results across sites, increasing microbiome diversity by increasing plant biomass, which created a shaded microclimate. This demonstration of consistent responses of microbiome diversity across a wide range of host species and environmental conditions suggests the possibility of a general, predictive understanding of microbiome diversity.

Pathogen and host genotype differently affect pathogen fitness through their effects on different life-history stages.

BACKGROUND: Adaptation of pathogens to their hosts depends critically on factors affecting pathogen reproductive rate. While pathogen reproduction is the end result of an intricate interaction between host and pathogen, the relative contributions of host and pathogen genotype to variation in pathogen life history within the host are not well understood. Untangling these contributions allows us to identify traits with sufficient genetic variation for selection to act and to identify mechanisms of coevolution between pathogens and their hosts. We investigated the effects of pathogen and host genotype on three life-history components of pathogen fitness; infection efficiency, latent period, and sporulation capacity, in the oat crown rust fungus, Puccinia coronata f.sp. avenae, as it infects oats (Avena sativa). RESULTS: We show that both pathogen and host genotype significantly affect total spore production but do so through their effects on different life-history stages. Pathogen genotype has the strongest effect on the early stage of infection efficiency, while host genotype most strongly affects the later life-history stages of latent period and sporulation capacity. In addition, host genotype affected the relationship between pathogen density and the later life-history traits of latent period and sporulation capacity. We did not find evidence of pathogen-by-host genotypic (GxG) interactions. CONCLUSION: Our results illustrate mechanisms by which variation in host populations will affect the evolution of pathogen life history. Results show that different pathogen life-history stages have the potential to respond differently to selection by host or pathogen genotype and suggest mechanisms of antagonistic coevolution. Pathogen populations may adapt to host genotypes through increased infection efficiency while their plant hosts may adapt by limiting the later stages of pathogen growth and spore production within the host.

Interactions between Fusarium verticillioides, Ustilago maydis, and Zea mays: an endophyte, a pathogen, and their shared plant host.

Highly diverse communities of microbial symbionts occupy eukaryotic organisms, including plants. While many well-studied symbionts may be characterized as either parasites or as mutualists, the prevalent but cryptic endophytic fungi are less easily qualified because they do not cause observable symptoms of their presence within their host. Here, we investigate the interactions of an endophytic fungus, Fusarium verticillioides with a pathogen, Ustilago maydis, as they occur within maize (Zea mays). We used experimental inoculations to evaluate metabolic mechanisms by which these three organisms might interact. We assessed the impacts of fungal-fungal interactions on endophyte and pathogen growth within the plant, and on plant growth. We find that F. verticillioides modulates the growth of U. maydis and thus decreases the pathogen's aggressiveness toward the plant. With co-inoculation of the endophyte with the pathogen, plant growth is similar to that which would be gained without the pathogen present. However, the endophyte may also break down plant compounds that limit U. maydis growth, and obtains a growth benefit from the presence of the pathogen. Thus, an endophyte such as F. verticillioides may function as both a defensive mutualist and a parasite, and express nutritional modes that depend on ecological context.

Metabolome and transcriptome of the interaction between Ustilago maydis and Fusarium verticillioides in vitro.

The metabolome and transcriptome of the maize-infecting fungi Ustilago maydis and Fusarium verticillioides were analyzed as the two fungi interact. Both fungi were grown for 7 days in liquid medium alone or together in order to study how this interaction changes their metabolomic and transcriptomic profiles. When grown together, decreased biomass accumulation occurs for both fungi after an initial acceleration of growth compared to the biomass changes that occur when grown alone. The biomass of U. maydis declined most severely over time and may be attributed to the action of F. verticillioides, which secretes toxic secondary metabolites and expresses genes encoding adhesive and cell wall-degrading proteins at higher levels than when grown alone. U. maydis responds to cocultivation by expressing siderophore biosynthetic genes and more highly expresses genes potentially involved in toxin biosynthesis. Also, higher expression was noted for clustered genes encoding secreted proteins that are unique to U. maydis and that may play a role during colonization of maize. Conversely, decreased gene expression was seen for U. maydis genes encoding the synthesis of ustilagic acid, mannosylerythritol D, and another uncharacterized metabolite. Ultimately, U. maydis is unable to react efficiently to the toxic response of F. verticillioides and proportionally loses more biomass. This in vitro study clarifies potential mechanisms of antagonism between these two fungi that also may occur in the soil or in maize, niches for both fungi where they likely interact in nature.

Network structure of resource use and niche overlap within the endophytic microbiome.

Endophytes often have dramatic effects on their host plants. Characterizing the relationships among members of these communities has focused on identifying the effects of single microbes on their host, but has generally overlooked interactions among the myriad microbes in natural communities as well as potential higher-order interactions. Network analyses offer a powerful means for characterizing patterns of interaction among microbial members of the phytobiome that may be crucial to mediating its assembly and function. We sampled twelve endophytic communities, comparing patterns of niche overlap between coexisting bacteria and fungi to evaluate the effect of nutrient supplementation on local and global competitive network structure. We found that, despite differences in the degree distribution, there were few significant differences in the global network structure of niche-overlap networks following persistent nutrient amendment. Likewise, we found idiosyncratic and weak evidence for higher-order interactions regardless of nutrient treatment. This work provides a first-time characterization of niche-overlap network structure in endophytic communities and serves as a framework for higher-resolution analyses of microbial interaction networks as a consequence and a cause of ecological variation in microbiome function.

In vitro interactions between Fusarium verticillioides and Ustilago maydis through real-time PCR and metabolic profiling.

The goal of this research was to determine mechanisms of interaction between endophytic strains of Fusarium verticillioides (Sacc.) Nirenberg and the pathogen, Ustilago maydis (DC) (Corda). Endophytic strains of the fungus F. verticillioides are commonly found in association with maize (Zea mays) and when co-inoculated with U. maydis, often lead to decreased disease severity caused by the pathogen. Here, we developed methods (liquid chromatography-mass spectrometry) to evaluate changes in relative concentration of metabolites produced during in vitro interactions between the endophyte and pathogen. Fungi were grown on two different media, in single and in confronted cultures. We used real-time PCR (qPCR) assays to measure relative changes in fungal biomass, that occurred in confronted cultures compared to single cultures. The results showed that most secondary metabolites are constitutively produced by each species. Metabolite profiles are complex for U. maydis (twenty chromatographic peaks detected) while relatively fewer compounds were detected for F. verticillioides (six chromatographic peaks). In confronted cultures, metabolite ratio (metabolite concentration/biomass) generally increases for U. maydis metabolites while no significant changes were observed for most F. verticillioides metabolites. The results show that F. verticillioides is a strong antagonist of U. maydis as its presence leads to large reductions in U. maydis biomass. We infer that few U. maydis metabolites likely serve antibiotic functions against F. verticillioides. The methods described here are sufficiently sensitive to detect small changes in biomass and metabolite concentration associated with differing genotypes of the interacting species.

Habitat-scale heterogeneity maintains fungal endophyte diversity in two native prairie legumes.

The assembly of fungal endophyte communities within plants depends on the complex interactions of fungal taxa, their host plants, and the abiotic environment. Prairie plant communities provide a unique avenue to explore the interplay of biotic and abiotic factors affecting endophyte communities, since the historical distribution of prairies spans a broad range of temperature and precipitation, while the distances between small fragments of contemporary prairie communities may challenge the dispersal capabilities of these otherwise ubiquitous fungi. We sampled foliar fungal endophytes from two native prairie legumes, purple and white prairie clovers (Dalea purpurea and D. candida), in 17 remnant prairie sites across Minnesota in order to evaluate the relative contributions of abiotic factors, host species, and dispersal limitation to the diversity and structure of these communities. We found that similarity of communities was significantly associated with their location along a temperature and precipitation gradient, and we showed a distance-decay relationship that suggests dispersal limitations only over very large spatial scales. Although the effect of host species was small relative to these other factors, the two Dalea species maintained distinct communities within sites where they co-occur. Our results illustrate the capacity of many of these endophyte taxa to disperse over large distances and across heterogeneous biotic and abiotic environments and suggest that the interplay of biotic and abiotic factors maintains high diversity observed in endophyte communities.

Plant diversity and litter accumulation mediate the loss of foliar endophyte fungal richness following nutrient addition.

Foliar fungal endophytes are ubiquitous plant symbionts that can affect plant growth and reproduction via their roles in pathogen and stress tolerance, as well as plant hormonal signaling. Despite their importance, we have a limited understanding of how foliar fungal endophytes respond to varying environmental conditions such as nutrient inputs. The responses of foliar fungal endophyte communities to increased nutrient deposition may be mediated by the simultaneous effects on within-host competition as well as the indirect impacts of altered host population size, plant productivity, and plant community diversity and composition. Here, we leveraged a 7-yr experiment manipulating nitrogen, phosphorus, potassium, and micronutrients to investigate how nutrient-induced changes to plant diversity, plant productivity, and plant community composition relate to changes in foliar fungal endophyte diversity and richness in a focal native grass host, Andropogon gerardii. We found limited evidence of direct effects of nutrients on endophyte diversity. Instead, the effects of nutrients on endophyte diversity appeared to be mediated by accumulation of plant litter and plant diversity loss. Specifically, nitrogen addition is associated with a 40% decrease in plant diversity and an 11% decrease in endophyte richness. Although nitrogen, phosphorus, and potassium addition increased aboveground live biomass and decreased relative Andropogon cover, endophyte diversity did not covary with live plant biomass or Andropogon cover. Our results suggest that fungal endophyte diversity within this focal host is determined in part by the diversity of the surrounding plant community and its potential impact on immigrant propagules and dispersal dynamics. Our results suggest that elemental nutrients reduce endophyte diversity indirectly via impacts on the local plant community, not direct response to nutrient addition. Thus, the effects of global change drivers, such as nutrient deposition, on characteristics of host populations and the diversity of their local communities are important for predicting the response of symbiont communities in a changing global environment.

Evolution in Candida albicans populations during a single passage through a mouse host.

The mechanisms and rates by which genotypic and phenotypic variation is generated in opportunistic, eukaryotic pathogens during growth in hosts are not well understood. We evaluated genomewide genetic and phenotypic evolution in Candida albicans, an opportunistic fungal pathogen of humans, during passage through a mouse host (in vivo) and during propagation in liquid culture (in vitro). We found slower population growth and higher rates of chromosome-level genetic variation in populations passaged in vivo relative to those grown in vitro. Interestingly, the distribution of long-range loss of heterozygosity (LOH) and chromosome rearrangement events across the genome differed for the two growth environments, while rates of short-range LOH were comparable for in vivo and in vitro populations. Further, for the in vivo populations, there was a positive correlation of cells demonstrating genetic alterations and variation in colony growth and morphology. For in vitro populations, no variation in growth phenotypes was detected. Together, our results demonstrate that passage through a living host leads to slower growth and higher rates of genomic and phenotypic variation compared to in vitro populations. Results suggest that the dynamics of population growth and genomewide rearrangement contribute to the maintenance of a commensal and opportunistic life history of C. albicans.