Contrasting carbon metabolism in saprotrophic and pathogenic microascalean fungi from Protea trees

Protea-associated Knoxdaviesia species grow on decaying inflorescences, yet are closely related to plant pathogens such as Ceratocystis albifundus. C. albifundus also infects Protea, but occupies a distinct niche. We investigated substrate utilization in two Knoxdaviesia saprotrophs, a generalist and a specialist, and the pathogen C. albifundus by integrating phenome and whole-genome data. On shared substrates, the generalist grew slightly better than its specialist counterpart, alluding to how it has maintained its Protea host range. C. albifundus grew on few substrates and had limited cell wall-degrading enzymes. It did not utilize sucrose, but may prefer soluble oligosaccharides. Nectar monosaccharides are likely important carbon sources for early colonizing Knoxdaviesia species. Once the inflorescence ages, they could switch to degrading cell wall components. C. albifundus likely uses its limited cell wall-degrading arsenal to gain access to plant cells and exploit internal resources. Overall, carbon metabolism and gene content in three related fungi reflected their ecological adaptations.     

Knoxdaviesia capensis: dispersal ecology and population genetics of a flower-associated fungus

Protea-associated fungi are dispersed between flower heads by mites, beetles and possibly birds. For the ophiostomatoid fungus, Knoxdaviesia proteae, these vectors offer regular dispersal between distant floral hosts. Unlike K. proteae, Knoxdaviesia capensis occupies multiple Protea host species. In this study, we aimed to determine whether the generalist K. capensis shares the long-distance dispersal pattern with specialist K. proteae and whether it moves freely between different host species. We evaluated the genetic structure of K. capensis from five populations of a wide-spread host and between sympatric hosts. Twelve K. capensis-specific microsatellite markers were developed and applied to 90 individuals. K. capensis showed high genetic diversity and almost maximal genotypic diversity. All populations were poorly differentiated, indicating the presence of long-distance dispersal. No differentiation could be detected between sympatric host populations, suggesting free dispersal between different hosts. This implies that the beetle and bird vectors that pollinate Protea species show the same non-specific movement.     

Unseen fungal biodiversity and complex inter-organismal interactions in Protea flower heads

A unique microbiome occurs within the flower heads of various Protea species endemic to Africa. These include two lineages of ophiostomatoid fungi, Knoxdaviesia (Microascales) and Sporothrix (Ophiostomatales), that have members occurring exclusively in this environment and that rely on mites as their primary mode of spore dissemination. The mites, in turn, attach to the bodies of Protea-pollinating beetles and the beaks and bodies of birds for long-distance movement, establishing a hierarchical dispersal network for the ophiostomatoid fungi. This inter-organismal network is highly successful, achieving fungal dispersal over vast distances. Multiple species of fungi, mites and bacteria have been described from this unique niche over the past four decades. The intricacies of their symbiotic interactions continue to be unravelled. This review covers all current knowledge of the “distinctly African” Protea-ophiostomatoid fungus environment and illustrates the depth of a fascinating unseen fungal biodiversity niche.     

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.     

Convergent evolution unites the population genetics of Protea-associated ophiostomatoid fungi

Knoxdaviesia and Sporothrix species occupy the flower heads of some Protea plants in southern Africa. Knoxdaviesia species display exceptional genetic diversity within the Core Cape Subregion (CCR) and are readily dispersed across large distances. This study aimed to determine whether overlapping ecologies have led to a similar population genetic structure in Sporothrix splendens. Two DNA sequence markers, β-tubulin and a microsatellite region, were amplified in 97 S. splendens strains from eight populations that span its host distribution. Genetic diversity was low in a geographically isolated population, but high elsewhere. CCR populations were closely related, showing isolation by distance with populations at the eastern edge of the sampling range. Like Knoxdaviesia species, long-distance dispersal of S. splendens spores is prevalent, although likely affected by patchy host populations. This study is the first to consider populations of a non-clinical Sporothrix species, providing insights into the population attributes of a naturally distributed species.     

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.     

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.

     

Development of polymorphic microsatellite markers for the genetic characterisation of Knoxdaviesia proteae (Ascomycota: Microascales) using ISSR-PCR and pyrosequencing

Knoxdaviesia proteae is one of the first native ophiostomatoid fungi discovered in South Africa, where it consistently occurs in the infructescences of the iconic Cape Biome plant, Protea repens. Although numerous studies have been undertaken to better understand the ecology of K. proteae, many questions remain to be answered, particularly given its unique niche and association with arthropods for dispersal. We describe the development and distribution of microsatellite markers in K. proteae through Interspersed Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) enrichment and pyrosequencing. A large proportion of the 31492 sequences obtained from sequencing the enriched genomic DNA were characterised by microsatellites consisting of short tandem repeats and di- and tri-nucleotide motifs. Seventeen percent of these microsatellites contained flanking regions sufficient for primer design. Twenty-three primer pairs were tested, of which 12 amplified and 10 generated polymorphic fragments in K. proteae. Half of these could be transferred to the sister species, K. capensis. The developed markers will be used to investigate the reproductive system, genetic diversity and dispersal strategies of K. proteae.     

A comparison of floral resource exploitation by native and invasive Argentine ants

Ants are often considered antagonists when they visit flowers because they typically steal nectar without providing pollination services. Previous research on ant–flower interactions on two species of South African Proteaceae in the Cape Floral Kingdom revealed that the invasive Argentine ant (Linepithema humile), but not native ants, displace other floral arthropod visitors. To determine how common Argentine ant use of inflorescences is, how Argentine and native ant visits differ in the numbers they recruit to inflorescences, and what factors may affect Argentine and native ant foraging in inflorescences, I surveyed 723 inflorescences in 10 species in the genera Protea and Leucospermum across 16 sites and compared ant presence and abundance in inflorescences with abundance at nearby cat food and jam baits. Argentine ants were the most commonly encountered ant of the 22 observed. Argentine ants, as well as six species of native ants were present in all inflorescences for which they were present at nearby baits. Mean Argentine ant abundance per inflorescence was 4.4 ± 0.84 (SE) ants and similar to that of Anoplolepis custodiens and Crematogaster peringueyi, but higher than observed for the other most commonly encountered native ants, Camponotus niveosetosus and Lepisiota capensis. Both Argentine ants and A. custodiens were more likely to be found foraging in spring and under humid conditions, and in inflorescences closer to the ground, with lower sucrose concentrations, and with a greater proportion of open flowers. Argentine ants were more likely to be found in Protea inflorescences, whereas A. custodiens and L. capensis more often visited Leucospermum inflorescences. Considering its displacement of floral arthropods and widespread use of Proteaceae inflorescences, the Argentine ant could be posing a serious threat to plant and pollinator conservation in this biodiversity hotspot.     

Comparative genome analysis of filamentous fungi reveals gene family expansions associated with fungal pathogenesis

Fungi and oomycetes are the causal agents of many of the most serious diseases of plants. Here we report a detailed comparative analysis of the genome sequences of thirty-six species of fungi and oomycetes, including seven plant pathogenic species, that aims to explore the common genetic features associated with plant disease-causing species. The predicted translational products of each genome have been clustered into groups of potential orthologues using Markov Chain Clustering and the data integrated into the e-Fungi object-oriented data warehouse (http://www.e-fungi.org.uk/). Analysis of the species distribution of members of these clusters has identified proteins that are specific to filamentous fungal species and a group of proteins found only in plant pathogens. By comparing the gene inventories of filamentous, ascomycetous phytopathogenic and free-living species of fungi, we have identified a set of gene families that appear to have expanded during the evolution of phytopathogens and may therefore serve important roles in plant disease. We have also characterised the predicted set of secreted proteins encoded by each genome and identified a set of protein families which are significantly over-represented in the secretomes of plant pathogenic fungi, including putative effector proteins that might perturb host cell biology during plant infection. The results demonstrate the potential of comparative genome analysis for exploring the evolution of eukaryotic microbial pathogenesis.     

Lifestyle Transitions in Fusarioid Fungi are Frequent and Lack Clear Genomic Signatures

The fungal genus Fusarium (Ascomycota) includes well-known plant pathogens that are implicated in diseases worldwide, and many of which have been genome sequenced. The genus also encompasses other diverse lifestyles, including species found ubiquitously as asymptomatic-plant inhabitants (endophytes). Here, we produced structurally annotated genome assemblies for five endophytic Fusarium strains, including the first whole-genome data for Fusarium chuoi. Phylogenomic reconstruction of Fusarium and closely related genera revealed multiple and frequent lifestyle transitions, the major exception being a monophyletic clade of mutualist insect symbionts. Differential codon usage bias and increased codon optimisation separated Fusarium sensu stricto from allied genera. We performed computational prediction of candidate secreted effector proteins (CSEPs) and carbohydrate-active enzymes (CAZymes)—both likely to be involved in the host–fungal interaction—and sought evidence that their frequencies could predict lifestyle. However, phylogenetic distance described gene variance better than lifestyle did. There was no significant difference in CSEP, CAZyme, or gene repertoires between phytopathogenic and endophytic strains, although we did find some evidence that gene copy number variation may be contributing to pathogenicity. Large numbers of accessory CSEPs (i.e., present in more than one taxon but not all) and a comparatively low number of strain-specific CSEPs suggested there is a limited specialisation among plant associated Fusarium species. We also found half of the core genes to be under positive selection and identified specific CSEPs and CAZymes predicted to be positively selected on certain lineages. Our results depict fusarioid fungi as prolific generalists and highlight the difficulty in predicting pathogenic potential in the group.     

Small RNAs from cereal powdery mildew pathogens may target host plant genes

Small RNAs (sRNAs) play a key role in eukaryotic gene regulation, for example by gene silencing via RNA interference (RNAi). The biogenesis of sRNAs depends on proteins that are generally conserved in all eukaryotic lineages, yet some species that lack part or all the components of the mechanism exist. Here we explored the presence of the RNAi machinery and its expression as well as the occurrence of sRNA candidates and their putative endogenous as well as host targets in phytopathogenic powdery mildew fungi. We focused on the species Blumeria graminis, which occurs in various specialized forms (formae speciales) that each have a strictly limited host range. B. graminis f. sp. hordei and B. graminis f. sp. tritici, colonizing barley and wheat, respectively, have genomes that are characterized by extensive gene loss. Nonetheless, we find that the RNAi machinery appears to be largely complete and expressed during infection. sRNA sequencing data enabled the identification of putative sRNAs in both pathogens. While a considerable part of the sRNA candidates have predicted target sites in endogenous genes and transposable elements, a small proportion appears to have targets in planta, suggesting potential cross-kingdom RNA transfer between powdery mildew fungi and their respective plant hosts.     

Ophiostoma quercus: An unusually diverse and globally widespread tree-infecting fungus

Ophiostoma quercus (Ascomycota, Ophiostomatales) is a globally widespread, insect-vectored fungus that colonizes a wide diversity of hardwood and conifer hosts. Although the fungus is considered to be non-pathogenic, it is closely related to the fungi that cause Dutch elm disease. We examined the global diversity of O. quercus based on a ribosomal RNA marker and three unlinked gene regions. The fungus exhibited substantial morphological diversity. In addition, O. quercus had high genetic diversity in every continent from which it was collected, although the fungus was most diverse in Eurasia. There was no evidence of geographical clustering of haplotypes based on phylogenetic and network analyses. In addition, the phylogenetic trees generated based on the different markers were non-congruent. These results suggest that O. quercus has been repeatedly moved around the globe, because of trade in wood products, and that the fungal species most likely outcrosses regularly. The high genetic diversity of the fungus, as well as its ability to utilize a wide variety of arthropod vectors and colonize a tremendous diversity of tree host species makes O. quercus truly unique among ophiostomatoid fungi.     

Initiation of Flowering in Protea compacta × Protea neriifolia Hybrid ‘Carnival’ Coincides with Expression of the FLOWERING LOCUS T Homologue

The Protea hybrid ‘Carnival’ (Protea compacta × Protea neriifolia) is responsive to seasonal change, arresting growth during the winter and producing flowers in the late summer after initiating flowering during the elongation of the spring flush. The large commercially attractive flower heads, consisting of over 200 florets, develop over a period of months. To begin to better understand the molecular factors that influence the transition to flowering in Protea, a homologue of the FLOWERING LOCUS T (FT) gene, ProFT, was isolated from ‘Carnival’, and its expression was analysed. ProFT showed increased expression in ‘Carnival’ leaves during October (13 h light/11 h dark; average daily temperature of 17 °C) at the time that floral organs were being pre-formed in the meristem. ProFT expression was fivefold higher in florally determined buds compared to that in leaves, and low levels were present in the vegetative meristems analysed. These results suggest that ProFT may act as a seasonally regulated floral inducer in ‘Carnival’, but based on spatial expression, data is also likely to play a role in inflorescence development and growth architecture.     

Genome-Wide Identification and Analysis of Genes Encoding Proteolytic Enzymes in Pineapple

Pineapple, Ananas comosus, is an economically important fruit crop. Recently its genome was completely sequenced and a total of 27,024 protein coding genes were predicted. Using a set of well evaluated bioinformatics tools we have predicted the protein subcellular locations and comparatively analyzed the protein conserved domains of the predicted proteomes in pineapple, Oryza sativa (rice), Sorghum bicolor (sorghum), and Brachypodium distachyson. Our analysis revealed that ~24–26 % of proteins were located in nucleus, 17–21 % in cytosol, 9–11 % in chloroplast, and 8–11 % proteins were secreted in these monocot plants. The secretomes in the four species were analyzed comparatively and a large number of secreted glycosyl hydrolases were identified. As pineapple proteolytic enzymes, knowns as bromelains, have been used for medical treatments, we focused on genome-wide identification and analysis of pineapple genes encoding proteases. A total of 512 pineapple genes encoding putative proteolytic enzymes were identified, with 152 secreted, 74 localized in cytosol, 67 in nucleus, 60 in chloroplast, 18 in mitochondria, and the remaining in other subcellular locations. The top large protease families in pineapple were papain family cysteine protease (62 genes), peptidase S8 family (56 genes), aspartyl protease family (38 genes), and serine carboxypeptidase (33 genes). Gene expression analysis revealed that among 512 protease genes 432 were expressed in various tissues and 72 genes were differentially expressed. The highly expressed protease genes were identified including 7 papain family cysteine proteases. The protease genes with the predicted protein subcellular locations will facilitate the efforts for examining their biological roles in pineapple growth and development and for expressing the recombinant proteases for medical use. The information of protein subcellular location of all plant species can be accessed at the PlantSecKB website (http://proteomics.ysu.edu/secretomes/plant.php).     

Defining the Predicted Protein Secretome of the Fungal Wheat Leaf Pathogen Mycosphaerella graminicola

The Dothideomycete fungus Mycosphaerella graminicola is the causal agent of Septoria tritici blotch, a devastating disease of wheat leaves that causes dramatic decreases in yield. Infection involves an initial extended period of symptomless intercellular colonisation prior to the development of visible necrotic disease lesions. Previous functional genomics and gene expression profiling studies have implicated the production of secreted virulence effector proteins as key facilitators of the initial symptomless growth phase. In order to identify additional candidate virulence effectors, we re-analysed and catalogued the predicted protein secretome of M. graminicola isolate IPO323, which is currently regarded as the reference strain for this species. We combined several bioinformatic approaches in order to increase the probability of identifying truly secreted proteins with either a predicted enzymatic function or an as yet unknown function. An initial secretome of 970 proteins was predicted, whilst further stringent selection criteria predicted 492 proteins. Of these, 321 possess some functional annotation, the composition of which may reflect the strictly intercellular growth habit of this pathogen, leaving 171 with no functional annotation. This analysis identified a protein family encoding secreted peroxidases/chloroperoxidases (PF01328) which is expanded within all members of the family Mycosphaerellaceae. Further analyses were done on the non-annotated proteins for size and cysteine content (effector protein hallmarks), and then by studying the distribution of homologues in 17 other sequenced Dothideomycete fungi within an overall total of 91 predicted proteomes from fungal, oomycete and nematode species. This detailed M. graminicola secretome analysis provides the basis for further functional and comparative genomics studies.     

Swimming with the giant: coexistence patterns of a new redfin minnow Pseudobarbus skeltoni from a global biodiversity hot spot

Ecological niche theory predicts that coexistence is facilitated by resource partitioning mechanisms that are influenced by abiotic and biotic interactions. Alternative hypotheses suggest that under certain conditions, species may become phenotypically similar and functionally equivalent, which invokes the possibility of other mechanisms, such as habitat filtering processes. To test these hypotheses, we examined the coexistence of the giant redfin Pseudobarbus skeltoni, a newly described freshwater fish, together with its congener Pseudobabus burchelli and an anabantid Sandelia capensis by assessing their scenopoetic and bionomic patterns. We found high habitat and isotope niche overlaps between the two redfins, rendering niche partitioning a less plausible sole mechanism that drives their coexistence. By comparison, environment–trait relationships revealed differences in species–environment relationships, making habitat filtering and functional equivalence less likely alternatives. Based on P. skeltoni's high habitat niche overlap with other species, and its large isotope niche width, we inferred the likelihood of differential resource utilization at trophic level as an alternative mechanism that distinguished it from its congener. In comparison, its congener P. burchelli appeared to have a relatively small trophic niche, suggesting that its trophic niche was more conserved despite being the most abundant species. By contrast, S. capensis was distinguished by occupying a higher trophic position and by having a trophic niche that had a low probability of overlapping onto those of redfins. Therefore, trophic niche partitioning appeared to influence the coexistence between S. capensis and redfins. This study suggests that coexistence of these fishes appears to be promoted by their differences in niche adaptation mechanisms that are probably shaped by historic evolutionary and ecological processes.