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.

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.

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.

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.

Genetic basis for high population diversity in Protea-associated Knoxdaviesia.

Sexual reproduction is necessary to generate genetic diversity and, in ascomycete fungi, this process is controlled by a mating type (MAT) locus with two complementary idiomorphs. Knoxdaviesia capensis and K. proteae (Sordariomycetes; Microascales; Gondwanamycetaceae) are host-specific saprophytic fungi that show high population diversity within their Protea plant hosts in the Cape Floristic Region of South Africa. We hypothesise that this diversity is the result of outcrossing driven by a heterothallic mating system and sought to describe the MAT1 loci of both species. The available genome assembly of each isolate contained only one of the MAT1 idiomorphs necessary for sexual reproduction, implying that both species are heterothallic. Idiomorph segregation during meiosis, a 1:1 ratio of idiomorphs in natural populations and mating experiments also supported heterothallism as a sexual strategy. Long-range PCR and shot-gun sequencing to identify the opposite idiomorph in each species revealed no sequence similarity between MAT1-1 and MAT1-2 idiomorphs, but the homologous idiomorphs between the species were almost identical. The MAT1-1 idiomorph contained the characteristic MAT1-1-1 and MAT1-1-2 genes, whereas the MAT1-2 idiomorph consisted of the genes MAT1-2-7 and MAT1-2-1. This gene content was similar to that of the three species in the Ceratocystidaceae (Microascales) with characterized MAT loci. The Knoxdaviesia MAT1-2-7 protein contained and alpha domain and predicted intron, which suggests that this gene arose from MAT1-1-1 during a recombination event. In contrast to the Ceratocystidaceae species, Knoxdaviesia conformed to the ancestral Sordariomycete arrangement of flanking genes and is, therefore, a closer reflection of the structure of this locus in the Microascalean ancestor.

Long-distance dispersal and recolonization of a fire-destroyed niche by a mite-associated fungus.

The Fynbos Biome in the Core Cape Subregion of South Africa is prone to recurrent fires that can clear vast areas of vegetation. Between periods of fire, ophiostomatoid fungi colonize the fruiting structures of serotinous Protea species through arthropod-mediated dispersal. Using microsatellite markers, this study considered the process whereby a Protea-associated ophiostomatoid fungus, Knoxdaviesia proteae, recolonizes a burnt area. The genetic diversity, composition and structure of fungal populations from young P. repens plants in a recently burnt area were compared to populations from the adjacent, unburnt Protea population. The only difference between K. proteae populations from the two areas was found in the number of private alleles, which was significantly higher in the unburnt population. The population structure, although weak, indicated that most K. proteae individuals from recently burnt areas originated from the unburnt population. However, individuals from unsampled source populations were also detected. This, together with the lack of isolation-by-distance across the landscape, suggested that long-distance dispersal is important for K. proteae to recolonize burnt areas. Similarly, the high level of gene flow and low differentiation observed between two distantly separated K. proteae populations also supported the existence of long-distance dispersal. The genetic cohesiveness of populations over long distances and the genetic diversity within populations could be attributed to frequent multiple fungal migration events mediated primarily by arthropods but, potentially, also by birds.

Evidence of novel plant-species specific ammonia oxidizing bacterial clades in acidic South African fynbos soils.

Ammonia-oxidizing bacteria (AOB) are essential in the biogeochemical cycling of nitrogen as they catalyze the rate-limiting oxidation of ammonia into nitrite. Since their first isolation in the late 19th century, chemolithoautotrophic AOBs have been identified in a wide range of natural (e.g., soils, sediments, estuarine, and freshwaters) and man created or impacted habitats (e.g., wastewater treatment plants and agricultural soils). However, little is known on the plant-species association of AOBs, particularly in the nutrient-starved fynbos terrestrial biome. In this study, we evaluated the diversity of AOBs in the plant canopy of three South African fynbos-specific plant species, namely Leucadendron xanthoconus, Leucospermum truncatulum and Leucadendron microcephalum, through the construction of amoA-gene clone libraries. Our results clearly demonstrate that plant-species specific and monophyletic AOB clades are present in fynbos canopy soils.

Soil bacteria hold the key to root cluster formation.

Root clusters are bunches of hairy rootlets that enhance nutrient uptake among many plants. Since first being reported in 1974, the involvement of rhizobacteria in their formation has received conflicting support. Attempts to identify specific causative organisms have failed and their role has remained speculative. We set up a gnotobiotic experiment using two root-clustered species, Viminaria juncea (Fabaceae) and Hakea laurina (Proteaceae), and inoculated them with two plant-growth-promoting rhizobacteria (PGPR), Bradyrhizobium elkanii and Bacillus mageratium, that produce indole-3-acetic-acid (IAA). Plants were suspended in water culture with four combinations of nitrogen and phosphorus. Clusters only developed in the presence of PGPR in two treatments, were greatly enhanced in another four, suppressed in five, and unaffected in five. Nitrogen amendment was associated with a higher density of clusters. Bradyrhizobium promoted cluster formation in Hakea, whereas Bacillus promoted cluster formation in Viminaria and suppressed it in Hakea. Greater root cluster numbers were due either to a larger root system induced by PGPR (indirect resource effect) and/or to more clusters per unit length of parent root (direct morphogenetic effect). The results are interpreted in terms of greater IAA production by Bradyrhizobium than Bacillus and greater sensitivity of Viminaria to IAA than Hakea.

Diversity of Penicillium section Citrina within the fynbos biome of South Africa, including a new species from a Protea repens infructescence.

During a survey of the fynbos biome in the Western Cape of South Africa, 61 Penicillium species were isolated and nine belong to Penicillium section Citrina. Based on morphology and multigene phylogenies, section Citrina species were identified as P. cairnsense, P. citrinum, P. pancosmium, P. pasqualense, P. sanguifluum, P. sizovae, P. sumatrense and P. ubiquetum. One of the species displayed unique phenotypic characters and DNA sequences and is described here as P. sucrivorum. Multigene phylogenies consistently resolved the new species in a clade with P. aurantiacobrunneum, P. cairnsense, P. miczynksii, P. neomiczynskii and P. quebecense. However, ITS, ß-tubulin and calmodulin gene sequences are unique for P. sucrivorum and growth rates on various media, the ability to grow at 30 C, a positive Ehrlich reaction and the absence of sclerotia on all media examined, distinguish P. sucrivorum from all of its close relatives.

Cryptococcus gattii in urban trees from cities in North-eastern Argentina.

In the city of Buenos Aires, Argentina, Cryptococcus gattii genotype AFLP4/VGI was found to be associated with decaying wood in hollows of different tree species. The aim of this study was to investigate the presence of C. gattii in the environment of riverside cities of the river Paraná, and to describe its serotypes and molecular types. Five hundred samples were collected in 50 parks by swabbing tree hollows. The samples were inoculated on caffeic acid agar supplemented with chloramphenicol, and incubated at 28 °C for 1 week with a daily observation. The isolates were identified by conventional methods. The serotype was determined by slide agglutination with specific antisera. Molecular typing was carried out by PCR-RFLP of the URA5 gene. Four isolates of C. gattii were recovered: Cryptococcus gattii serotype B, genotype AFLP4/VGI, isolated from Eucalyptus sp. in the city of Rosario and from Grevillea robusta in the city of La Paz; and C. gattii serotype C, genotype AFLP5/VGIII, isolated from two different Tipuana tipu trees in the city of Resistencia. Here, we report for the first time the isolation of C. gattii serotype C, genotype AFLP5/VGIII, from environmental samples in Argentina.

Biotic and abiotic constraints that facilitate host exclusivity of Gondwanamyces and Ophiostoma on Protea.

Estimations of global fungal diversity are hampered by a limited understanding of the forces that dictate host exclusivity in saprobic microfungi. To consider this problem for Gondwanamyces and Ophiostoma found in the flower heads of Protea in South Africa, we determined the role of various factors thought to influence their host exclusivity. Results showed that various biotic and abiotic factors influence the growth and survival of these fungi in vitro. Monitoring temperature and relative humidity (RH) fluctuations within infructescences in vivo revealed considerable microclimatic differences between different Protea spp. Fungal growth and survival at different RH levels experienced in the field suggested that this factor does not play a major role in host exclusivity of these fungi. Maximum temperatures within infructescences and host preferences of the vectors of Gondwanamyces and Ophiostoma appear to play a substantial part in determining colonisation of Protea in general. However, these factors did not explain host exclusivity of specific fungal species towards particular Protea hosts. In contrast, differential growth of fungal species on media containing macerated tissue of Protea showed that Gondwanamyces and Ophiostoma grow best on tissue from their natural hosts. Thus, host chemistry plays a role in host exclusivity of these fungi, although some species grew vigorously on tissue of Protea spp. with which they are not naturally associated. A combination of host chemistry and temperature partially explains host exclusivity, but the relationship for these factors on the tested saprobic microfungi and their hosts is clearly complex and most likely includes combinations of various biotic and abiotic factors including those emerging from this study.

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.

Mite-mediated hyperphoretic dispersal of Ophiostoma spp. from the infructescences of South African Protea spp.

Ophiostomatoid fungi are well known as economically important pathogens and agents of timber degradation. A unique assemblage of these arthropod-associated organisms including species of Gondwanamyces G. J. Marais and M. J. Wingf., and Ophiostoma Syd. and P. Syd. occur in the floral heads (infructescences) of Protea L. species in South Africa. It has recently been discovered that Ophiostoma found in Protea flower-heads are vectored by mites (Acarina) including species of: Tarsonemus Canestrini and Fonzago, Proctolaelaps Berlese, and Trichouropoda Berlese. It is, however, not known how the mites carry the fungi between host plants. In this study, we consider two possible modes of mite dispersal. These include self-dispersal between infructescences and dispersal through insect vectors. Results showed that, as infructescences desiccate, mites self-disperse to fresh moist infructescences. Long-range dispersal is achieved through a phoretic association with three beetle species: Genuchus hottentottus (F.), Trichostetha fascicularis L., and T. capensis L. The long-range, hyperphoretic dispersal of O. splendens G. J. Marais and M. J. Wingf. and O. phasma Roets et al. seemed effective, because their hosts were colonized during the first flowering season 3-4 yr after fire.

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.

Heteroconium and Pirozynskiella n. gen., with comments on conidium transseptation.

Heteroconium citharexyli, the type species of this genus, is illustrated and redescribed as a sooty mold bearing acropetal chains of conidia showing a basifugal sequence of septation. Heteroconium neriifoliae, H. glutinosa and the Heteroconium synanamorph of Antennulariella concinna are congeneric. The latter species is neotypified, illustrated and described. Pirozynskiella new genus, typified by P. solaninum comb. nov. (-Helminthosporium solaninum), differs from Heteroconium in having an obligate association with asterinaceous fungi and in the centrifugal sequence of conidium transseptation after the initial median septum. Pirozynskiella costaricensis comb. nov. (-Dendryphion costaricensis) is illustrated and described. Heteroconium tetracoilum and H. chaetospira are fungicoles of wood and bark; the former has basifugal conidium septation and the latter has a centrifugal sequence. The two latter species can be excluded from the Heteroconium. Basifugal and centrifugal septation of conidia is discussed with reference to several sooty molds, to some foliicolous anamorphs with subcuticular hyphae (Heterosporiopsis) and to some wood and bark hyphomycetes (Septonema, Taeniolella). Ten other species included in Heteroconium are known to me only from their original descriptions; only one (H. asiaticum) is probably a sooty mold.

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.

Diversity and distribution of saprobic microfungi in leaf litter of an Australian tropical rainforest.

The diversity and distribution of microfungal assemblages in leaf litter of a tropical Australian forest was assessed using two methods: (1) cultures were isolated using a particle filtration protocol (wet season 2001), and (2) fruit bodies were observed directly on leaf surfaces following incubation in humid chambers (wet and dry season of 2002). Four tree species were studied using both methods, namely Cryptocarya mackinnoniana (Lauraceae), Elaeocarpus angustifolius (Elaeocarpaceae), Ficus pleurocarpa (Moraceae), and Opisthiolepis heterophylla (Proteaceae). An additional two species, Darlingia ferruginea (Proteaceae) and Ficus destruens (Moraceae), were studied using direct observations. In total, fruiting bodies of 185 microfungal species were recorded on leaf surfaces (31-81 species per tree species), and 419 morphotypes were detected among isolates obtained by particle filtration (111-203 morphotypes per tree species). Although the observed microfungal diversity was higher with the particle filtration protocol, both methods concurred with respect to microfungal distributions. The overlap of microfungal species in pair wise comparisons of tree species was low (14-30%), and only 2 and 3% of microfungal species were observed in leaves of all tree species by particle filtration and by direct observations respectively. Multivariate analysis of data from direct observations confirmed the hypothesis that microfungal assemblages are strongly influenced by host phylogeny and are also affected by seasonal and site factors. The importance of host species in shaping microfungal distributions was also supported by the particle filtration data. Several taxa new to science, as well as some widespread saprotrophs, were detected on only one host. The underlying reasons for this affinity remain unclear, but we hypothesise that a number of factors may be involved such as fungal adaptation to plant secondary metabolites or the presence of a biotrophic phase in the fungus' life cycle.

Bacterial diversity in the rhizosphere of Proteaceae species.

The Cape Floral Kingdom is an area of unique plant biodiversity in South Africa with exceptional concentrations of rare and endemic species and experiencing drastic habitat loss. Here we present the first molecular study of the microbial diversity associated with the rhizosphere soil of endemic plants of the Proteaceae family (Leucospermum truncatulum and Leucadendron xanthoconus). Genomic DNA was extracted from L. truncatulum rhizosphere soil, L. xanthoconus rhizosphere and non-rhizosphere soil and used as a template for the polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA gene (rDNA). Construction and sequencing of 16S rDNA libraries revealed a high level of biodiversity and led to the identification of several novel bacterial phylotypes. The bacterial community profiles were compared by 16S rDNA denaturing gradient gel electrophoresis (DGGE). Cluster analysis and biodiversity indices revealed that the rhizosphere soil samples were more similar to each other than to non-rhizosphere soil and the rhizosphere soil contained a bacterial diversity that was richer and more equitable compared with non-rhizosphere soil. A Chloroflexus and an Azospirillum genospecies were restricted to the L. xanthoconus rhizosphere soil and Stenotrophomonas genospecies was identified in all rhizosphere soil samples but was not present in the non-rhizosphere soil. Taxon-specific nested PCR and DGGE-identified differences between the Proteaceae plant rhizosphere soil with a Frankia genospecies restricted the L. truncatulum rhizosphere. Archaea-specific rDNA PCR, DGGE and DNA sequencing revealed that Crenarcheote genospecies were excluded from the plant rhizosphere soil and only present in non-rhizosphere soil.