Metagenomics of Toenail Onychomycosis in Three Victorian Regions of Australia.

Onychomycosis is a fungal disease of the nail that is found worldwide and is difficult to diagnose accurately. This study used metagenomics to investigate the microbiology of 18 clinically diagnosed mycotic nails and two normal nails for fungi and bacteria using the ITS2 and 16S loci. Four mycotic nails were from Bass Coast, six from Melbourne Metropolitan and eight from Shepparton, Victoria, Australia. The mycotic nails were photographed and metagenomically analysed. The ITS2 sequences for T. rubrum and T. interdigitale/mentagrophytes averaged over 90% of hits in 14/18 nails. The high abundance of sequences of a single dermatophyte, compared to all other fungi in a single nail, made it the most likely infecting agents (MLIA). Trichophyton rubrum and T. interdigitale/mentagrophytes were found in Bass Coast and Shepparton while only T. interdigitale/mentagrophytes was found in Melbourne. Two nails with T. interdigitale/mentagrophytes mixed with high abundance non-dermatophyte moulds (NDMs) (Aspergillus versicolor, Acremonium sclerotigenum) were also observed. The two control nails contained chiefly Fusarium oxysporum and Malassezia slooffiae. For bacteria, Staphylococcus epidermidis was in every nail and was the most abundant, including the control nails, with an overall mean rate of 66.01%. Rothia koreensis, Corynebacterium tuberculostearicum, and Brevibacterium sediminis also featured.

Enhanced Antimicrobial Activity and Low Phytotoxicity of Acoustically Synthesized Large Aspect Ratio Cu-BTC Metal-Organic Frameworks with Exposed Metal Sites.

Metal-organic frameworks (MOFs) have recently been shown to be effective antimicrobial agents, particularly if they comprise pathogenicidal metal ions. Nevertheless, the accessibility of these active metal sites to the pathogen, and hence the MOFs' antimicrobial activity itself, is often poor since the metal nodes are usually embedded deep within its three-dimensional (3D) structure. We show that a unique copper-based (copper(II)-benzene-1,3,5-tricarboxylate) MOF, whose quasi-two-dimensional (quasi-2D) swordlike structure facilitates exposure of the metal ions along its surface, exhibits enhanced antimicrobial properties against three representative plant pathogens: a bacterium (Pseudomonas syringae), a fungus (Fusarium solani), and a virus (Odontoglossum ringspot virus (ORSV)). Such superior antimicrobial activity results in low minimum inhibitory concentrations (MICs)─half that of a commercial pesticide and an eighth of its conventional 3D cubic MOF counterpart (HKUST-1)─and hence low phytotoxicity, which can be attributed to the accessibility of the surface copper sites to the pathogen, thereby facilitating their adhesion and physical contact with the MOF. Additionally, we observed that orchids treated with the quasi-2D MOF showed negligible phytotoxicity and 80% decreased viral load. This work constitutes the first study to demonstrate the antimicrobial properties of this novel MOF against bacterial, fungal, and viral plant pathogens, and the first chemical control of ORSV.

Why Variation in Flower Color May Help Reproductive Success in the Endangered Australian Orchid Caladenia fulva.

Caladenia fulva G.W. Carr (Tawny Spider-orchid) is a terrestrial Australian endangered orchid confined to contiguous reserves in open woodland in Victoria, Australia. Natural recruitment is poor and no confirmed pollinator has been observed in the last 30 years. Polymorphic variation in flower color complicates plans for artificial pollination, seed collection and ex situ propagation for augmentation or re-introduction. DNA sequencing showed that there was no distinction among color variants in the nuclear ribosomal internal transcribed spacer (ITS) region and the chloroplast trnT-trnF and matK regions. Also, authentic specimens of both C. fulva and Caladenia reticulata from the reserves clustered along with these variants, suggesting free interbreeding. Artificial cross-pollination in situ and assessment of seed viability further suggested that no fertility barriers existed among color variants. Natural fruit set was 15% of the population and was proportional to numbers of the different flower colors but varied with orchid patch within the population. Color modeling on spectral data suggested that a hymenopteran pollinator could discriminate visually among color variants. The similarity in fruiting success, however, suggests that flower color polymorphism may avoid pollinator habituation to specific non-rewarding flower colors. The retention of large brightly colored flowers suggests that C. fulva has maintained attractiveness to foraging insects rather than evolving to match a scarce unreliable hymenopteran sexual pollinator. These results suggest that C. fulva should be recognized as encompassing plants with these multiple flower colors, and artificial pollination should use all variants to conserve the biodiversity of the extant population.

Assessing the Plant Growth Promoting and Arsenic Tolerance Potential of Bradyrhizobium japonicum CB1809.

Accumulation of heavy metals in soil is of concern to the agricultural production sector, because of the potential threat to food quality and quantity. Inoculation with plant growth-promoting bacteria (PGPR) has previously been shown to alleviate heavy metal stress but the mechanisms are unclear. Potential mechanisms by which inoculation with Bradyrhizobium japonicum CB1809 affected the legume soybean (Glycine max cv. Zeus) and the non-legume sunflower (Helianthus annus cv. Hyoleic 41) were investigated in solution culture under 5 µM As stress. Adding As resulted in As tissue concentrations of up to 5 mg kg-1 (shoots) and 250 mg kg-1 (roots) in both species but did not reduce shoot or root biomass. Inoculation increased root biomass but only in the legume (soybean) and only with As. Inoculation resulted in large (up to 100%) increases in siderophore concentration but relatively small changes (±10-15%) in auxin concentration in the rhizosphere. However, the increase in siderophore concentration in the rhizosphere did not result in the expected increases in tissue N or Fe, especially in soybean, suggesting that their function was different. In conclusion, siderophores and auxins may be some of the mechanisms by which both soybean and sunflower maintained plant growth in As-contaminated media.

Predominance of Trichophyton interdigitale Revealed in Podiatric Nail Dust Collections in Eastern Australia.

Toenail onychomycosis caused by dermatophytes is a significant medical and financial worldwide burden. Relatively scant research has been undertaken as to the predominant species and strains causing this condition in Australia, which is a unique isolated continent with diverse geographical, climatic and population regions. Four regions were selected in Eastern Australia: Far North Queensland, Rural Victoria, Melbourne Metropolitan and Tasmania. From each of these areas, communal nail dust bags from podiatric physicians' work were collected and analysed. A total of 32 dust bags were collected: 10 from Far North Queensland, 8 from Melbourne Metropolitan, 8 from Rural Victoria and 6 from Tasmania. Dermatophyte test medium was used to isolate dermatophytes from the dust, and the colonies were subcultured to Potato Dextrose Agar. Of the bags collected, in total 69% were positive for dermatophytes: 40% from Far North Queensland, 75% from Melbourne Metropolitan, 88% from Rural Victoria and 83% from Tasmania. The internal transcribed spacer (ITS) region of ribosomal DNA was used to identify and compare isolates. A total of 148 dermatophyte strains were identified. The predominant species isolated was Trichophyton interdigitale (125 isolates), which was found in all four regions. This species was further subdivided into four ITS genotypes: the first two were present in all regions, but the third was found only in the Melbourne Metropolitan area and the fourth only in Tasmania. Only one strain of Trichophyton rubrum was found and only in Rural Victoria. Eighteen isolates of Arthroderma quadrifidum were cultured from Rural Victoria and Tasmania and were further classified into three ITS genotypes. Some isolates rarely reported in clinical material were identified as Paraphyton cookei, Arthroderma tuberculatum and Arthroderma crocatum. A potentially new species of Arthroderma was also found in Melbourne Metropolitan. These findings reveal a unique dermatophyte fingerprint in toenails for Eastern Australia.

Matching symbiotic associations of an endangered orchid to habitat to improve conservation outcomes.

Background and Aims: An understanding of mycorrhizal variation, orchid seed germination temperature and the effect of co-occurring plant species could be critical for optimizing conservation translocations of endangered plants with specialized mycorrhizal associations. Methods: Focusing on the orchid Thelymitra epipactoides, we isolated mycorrhizal fungi from ten plants within each of three sites; Shallow Sands Woodland (SSW), Damp Heathland (DH) and Coastal Heathland Scrub (CHS). Twenty-seven fungal isolates were tested for symbiotic germination under three 24 h temperature cycles: 12 °C for 16 h-16 °C for 8 h, 16 °C for 16 h-24 °C for 8 h or 27 °C constant. Fungi were sequenced using the internal transcribed spacer (ITS), nuclear large subunit 1 (nLSU1), nLSU2 and mitochondrial large rRNA gene (mtLSU). Orchids were grown to maturity and co-planted with each of ten associated plant species in a glasshouse experiment with tuber width measured at 12 months after co-planting. Key Results: Two Tulasnella fungal lineages were isolated and identified by phylogenetic analyses, operational taxonomic unit 1 (OTU1) and 'T. asymmetrica'. Fungal lineages were specific to sites and did not co-occur. OTU1 (from the SSW site) germinated seed predominantly at 12-16 °C (typical of autumn-winter temperature) whereas 'T. asymmetrica' (from the DH and CHS sites) germinated seed across all three temperature ranges. There was no difference in the growth of adult orchids germinated with different OTUs. There was a significant reduction in tuber size of T. epipactoides when co-planted with six of the commonly co-occurring plant species. Conclusions: We found that orchid fungal lineages and their germination temperature can change with habitat, and established that translocation sites can be optimized with knowledge of co-occurring plant interactions. For conservation translocations, particularly under a changing climate, we recommend that plants should be grown with mycorrhizal fungi tailored to the recipient site.

Peptide-mediated cell penetration and targeted delivery of gold nanoparticles into lysosomes.

There is considerable interest in the sub-cellular targeting and delivery of biomolecules, therapeutic and imaging agents, and nanoparticles and nanoparticle conjugates into organelles for therapeutic and imaging purposes. To date, a number of studies have used sorting peptides for targeted delivery of cargo into different cell organelles but not into lysosomes. In this study, the delivery of 13-nm gold nanoparticles across the cell membrane followed by targeted localisation into the lysosomes of a mammalian cell line was examined using novel combinations of cell-penetrating peptides and lysosomal sorting peptides conjugated to the nanoparticles. Using a combination of fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy techniques, we show that these nanoconjugates were efficiently and selectively delivered into the lysosomes with minimal cytotoxic effects. This novel targeted delivery system may underpin the development of a new strategy for the treatment of lysosomal storage diseases by exploiting the large surface area of nanoparticles to deliver drugs or replacement enzymes directly to the lysosomes.

Functional and genetic diversity of mycorrhizal fungi from single plants of Caladenia formosa (Orchidaceae).

BACKGROUND AND AIMS: Mycorrhizal associations are essential to the plant kingdom. The largest flowering plant family, the Orchidaceae, relies on mycorrhizal fungi for germination, growth and survival. Evidence suggests varying degrees of fungal-host specificity based on a single fungal isolate from a single plant. This paper shows for the first time the diversity of endophytes colonizing in a single plant over consecutive years and the functional significance of this diversity. METHODS: Stem-collars of Caladenia formosa were collected in different seasons and years. Mycorrhizal fungi isolated were tested for their efficacy to induce leafing and genetically determined using ITS-RFLP and sequencing. RESULTS: Multiple mycorrhizal fungi were repeatedly isolated from a single collar that displayed varying effectiveness in germination percentages and adult leaf length. Additional factors contributed to the isolation of effective mycorrhizal fungi; fungal collection season, year of collection and individual isolates. Surface sterilization only improved the number of isolated mycorrhizal fungi. Dual inoculation did not increase germination. All 59 mycorrhizal fungi effective in germinating seed belonged to one clearly defined ITS (internal transcribed spacer) clade and clustered close to Sebacina vermifera (79-89 % homology). Isolates resulting in the greatest germination were not necessarily those resulting in the greatest survival and growth 1 year after germination. CONCLUSION: Single orchid plants contained multiple mycorrhizal fungal strains of one species that had diverse functional differences. These results suggest that our current knowledge of fungal-host specificity may be incomplete due to experimental and analytical limitations. It also suggests that the long-term effectiveness of a mycorrhizal fungus or fungi could only be found by germination and longer-term growth tests rather than genetically.

Cloning and sequence characterization of a non-reducing polyketide synthase gene from the lichen Xanthoparmelia semiviridis.

Lichens produce a diverse array of secondary metabolites that have shown various biological activities. Of particular interest are the coupled phenolics that originate from polyketide pathways, such as depsides, depsidones and usnic acids, which are produced almost solely by lichens. Based on the presumed catalytic domains required for the synthesis of the key intermediates beta-orsellinic acid and methylphloroacetophenone, two pairs of degenerate primers were designed to target specifically the beta-ketoacylsynthase (KS) and C-methyltransferase (CMeT) domains of fungal non-reducing polyketide synthase (NR-PKS) genes with CMeT domains. These primers were used to explore the genome of the lichen Xanthoparmelia semiviridis, which produces beta-orcinol depsidones and usnic acid. One of the two KS domains amplified from genomic DNA of field-collected X. semiviridis was used as a probe to recover the candidate PKS gene. A 13 kb fragment containing an intact putative PKS gene (xsepks1) of 6555 bp was recovered from a partial genomic library. The inferred amino acid sequence indicated that xsepks1 encodes a protein of 2164 amino acids and contains KS, acyltransferase (AT), acyl carrier protein (ACP) and CMeT domains as expected. This demonstrated a successful strategy for targeting non-reducing PKS genes with CMeT domains. Integration of the 5' fragment of xsepks1, including the native promoter, into Aspergillus nidulans by cotransformation resulted in the transcription of the 5'xsepks1 and the splicing of a 63 bp intron, suggesting that A. nidulans could be a suitable heterologous host for xsepks1 expression.

Genetic variation within Podosphaera tridactyla reveals a paraphyletic species complex with biological specialization towards specific Prunus subgenera.

Podosphaera tridactyla (Ascomycota: Erysiphales) is a morphologically variable species occurring on Prunus s. lat. In order to assess the genetic variation within this species, the rDNA ITS region was amplified from 29 specimens from a range of Prunus species collected in Australia, Switzerland, and Korea. RFLP analysis of the PCR products revealed six groups, and a comparison of sequences from representatives of these groups revealed three clades: Clade I contained all specimens from Prunus subgen. Prunus; Clade 2 specimens from a variety of Prunus subgenera, except subgen. Prunus, and Podosphaera longiseta was closely allied to this clade; and Clade 3 contained two specimens, from Japan and Korea. Phylogenetic analysis comparing P. tridactyla with of a range of Podosphaera species suggests that P. tridactyla is paraphyletic.

A specific primer PCR and RFLP assay for the rapid detection and differentiation in planta of some Mycosphaerella species associated with foliar diseases of Eucalyptus globulus.

It is difficult to accurately identify Mycosphaerella species associated with leaf diseases of Eucalyptus based on morphological characters, as there is considerable overlap between very similar species and subspecies, and isolation from the host is not easy. Thus, a PCR and RFLP assay based on the ITS region of nr DNA was developed for the rapid detection and differentiation of M. nubilosa, M. cryptica and two non-sporing unidentified Mycosphaerella species isolated from the foliage of trees in resistant and susceptible families of E. globulus in a seed orchard at Kinglake West, Victoria, Australia. The M. nubilosa primer pair MNF/MNR was highly specific. A PCR-RFLP system based on the primer pair MCF/MCR, coupled with two restriction enzymes (DdeI and Tru1 I), differentiated M. cryptica, M. nubilosa, M. tasmaniensis and M. aff. vespa. One of the unidentified field-isolated Mycosphaerella species was identified as M. grandis on the basis of ITS sequence data while the other species remains unidentified. A PCR-RFLP system based on the primer pair U1F/U1R, coupled with the restriction enzyme StyI, differentiated between the two unidentified species. Unexpectedly, unlike isolation and culture studies, these assays detected M. nubilosa, M. cryptica and M. grandis in all single lesions examined on both juvenile and adult leaves, and on both highly resistant and highly susceptible E. globulus trees at this site.