Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

Fungal microparasites (here chytrids) are widely distributed and yet, they are often overlooked in aquatic environments. To facilitate the detection of microparasites, we revisited the applicability of two fungal cell wall markers, Calcofluor White (CFW) and wheat germ agglutinin (WGA), for the direct visualization of chytrid infections on phytoplankton in laboratory-maintained isolates and field-sampled communities. Using a comprehensive set of chytrid–phytoplankton model pathosystems, we verified the staining pattern on diverse morphological structures of chytrids via fluorescence microscopy. Empty sporangia were stained most effectively, followed by encysted zoospores and im-/mature sporangia, while the staining success was more variable for rhizoids, stalks, and resting spores. In a few instances, the staining was unsuccessful (mostly with WGA), presumably due to insufficient cell fixation, gelatinous cell coatings, and multilayered cell walls. CFW and WGA staining could be done in Utermöhl chambers or on polycarbonate filters, but CFW staining on filters seemed less advisable due to high background fluorescence. To visualize chytrids, 1 µg dye mL−1 was sufficient (but 5 µg mL−1 are recommended). Using a dual CFW–WGA staining protocol, we detected multiple, mostly undescribed chytrids in two natural systems (freshwater and coastal), while falsely positive or negative stained cells were well detectable. As a proof-of-concept, we moreover conducted imaging flow cytometry, as a potential high-throughput technology for quantifying chytrid infections. Our guidelines and recommendations are expected to facilitate the detection of chytrid epidemics and to unveil their ecological and economical imprint in natural and engineered aquatic systems.

Molecular Diagnosis of Fungal Sinusitis Using Limit of Detection

Background: As an inflammatory process that involves the paranasal sinuses, chronic sinusitis (CS) is one of the most prevalent chronic illnesses that affects all age groups. Parasitic fungi are involved in sinusitis infections. Objective: This study is aimed at the molecular detection of sinusitis caused by such fungi. Methods: Seventy-two samples were collected from the secretions of maxillary and frontal sinuses of patients from Rasoul-e Akram (PbUH) Hospital in Tehran during sinus operation. Fungal genomic DNA was extracted by a DNP kit. The detection of fungi was carried out by employing a sequence-specific target, namely mt cyte b gene locus, and using primers. Polymerase chain reaction (PCR) was optimized, and the limit of detection (LOD) and specificity tests were performed. The amplicon was cloned by the T/A cloning method, which was used for sequencing and positive control. Results: The 430-bp PCR product underwent appropriate propagation before being amplified and was observed on 1.5% electrophoreses gel. The evaluation of the selected primers with seven DNA constructs from another microorganisms demonstrated 100% specificity. The limit of detection of the optimized test was evaluated up to 50 fungi. Out of 72 samples, 9.7% were positive for fungi existence. Conclusions: This study indicated that molecular diagnosis of the target mt cyte b gene using LOD enhances clinical laboratory detection of fungal sinusitis.

Morphological and spectroscopic analysis of snow and glacier algae and their parasitic fungi on different glaciers of Svalbard

The results show the morphological analyses and spectroscopic studies of snow and glacier algae and their parasitic fungi in Svalbard (High Arctic). Fixed algal cells of two species, Sanguina nivaloides and Ancylonema nordenskioeldii, were imaged using light microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Fluorescence microscopy using Calcofluor white stain supported the observations of parasitic fungi on the algal cells. Images in brightfield microscopy showed chytrid-like fungi penetrating the cells of both algal species. Parasites were found to colonize the cells of A. nordenskioeldii and hypnozygotes of S. nivaloides, while no fungi infected the cyst stages of S. nivaloides. The autofluorescence analysis revealed the ability of S. nivaloides to glow when excited with different wavelengths, while A. nordenskioeldii did not fluoresce. The hypnozygotes of S. nivaloides emitted brighter fluorescence than the cysts, and the most intense luminosity was observed in the UV range. The Fourier-transform infrared spectroscopy (FTIR) and energy-dispersive X-ray spectroscopy (EDS) spectroscopic analysis showed differences in the chemical composition between samples collected from three different sites. Samples dominated by cyst cells were characterized by the presence of an abundant polysaccharide envelope.

Ethno pharmacological Efficiency of Andrographis Paniculata against Tuber Rot Disease of Manihot Esculenta (Cassava)

Cassava is a chief and energy rich carbohydrates food, which might be the reason many parasitic fungi adhere within the plant that causes huge damage to the crop during the post harvesting stage or storage period. In the present study, the ethno-based possible eradication of parasitic fungi was highlighted. Andrographis paniculata Burm. f. Nees and Stachytarpheta indica were chosen for its popular medicinal values and the phytochemical contents were characterized. Its potency showed an inhibitory effects against tuber rot disease causing fungal pathogens viz., Rhizopus sp, Mucor sp., Aspergillus niger, Aspergillus flavus and Fusarium oxysporum in Cassava by disc – diffusion methods. The diameter of the zone of inhibition at various concentrations (2.5, 5, 7.5 and 10 w/v) of leaf extracts was observed with the maximum in A. paniculata (20mm with 10 w/v conc.) against Fusarium oxysporum followed by A. niger (17mm at 10w/v conc.). The preliminary phytochemical screening of Andrographis paniculata revealed the presence of phytoconstituents like alkaloids, flavonoids, phenols, and tannins. In GCMS analysis, Benzene,1,2,3-trimethoxy-5-(2-propenyl)-, Cyclopenta [c] pyran-4-carboxylic acid, 7-methyl-, methyl ester and 9,12,15-Octadecatrienoic acid, methyl ester, (Z, Z, Z)- covered the high areas, that might be responsible or can possess the antifungal activity against Tuber root rot causing fungal pathogens. Furthermore, extensive studies are recommended to characterize each compound in the crude extracts of A. paniculata to enhance or to acclaim remedy for various diseases in plants. This study reveals the acknowledgement of the medicinal plant A. paniculata for its remarkable organic or eco-friendly fungicide potential against cassava tuber rot diseases.

Advances in understanding the biology and epidemiology of rust diseases of cereals

Rust fungi (order: Pucciniales) constitute the largest group of plant parasitic fungi and include many species of agricultural importance. This includes the three wheat rust fungi (Puccinia graminis f. sp. tritici, Puccinia striiformis f. sp. tritici and Puccinia triticina) that have posed a threat to crop production throughout history. This chapter provides an overview of the wheat rust pathogen lifecycle that has been critical to the design of effective disease management strategies and discusses recent integration of basic biological knowledge and genomic-led tools within an epidemiological framework. Furthermore, we include a case study on the “field pathogenomics” technique, illustrating the value of genomic-based tools in disease surveillance activities. Bringing together advances in understanding basic pathogen biology, developments in modelling for disease forecasting and identification, alongside genomic-led advances in surveillance and resistance gene cloning, holds great promise for curtailing the threat of these notorious pathogens.

Superficial Dermatophytic Infection Prevention and Its Management: A Review

Dermatophytes are a group of parasitic fungi that live at the expense of the keratin in the skin, nails and hair. They are generally confined to the stratum corneum of the epidermis and skin appendages, particularly in the tropics, where heat and humidity provide the ideal conditions for the growth of fungi that cause cutaneous infections. The dermatophytes that usually cause only superficial infections of the skin are grouped into three genera: Trichophyton, Microsporum, and Epidermophyton. They can be classified into three groups according to their normal habitats: anthropophilic species, zoophilic species and geophilic species. Dermatophyte infections are subclassified in Latin names according to the sites of skin involved, e.g. Tinea faciei, Tinea manuum, Tinea corporis, Tinea cruris, Tinea pedis, Tinea capitis, Tinea unguium. Trichophyton rubrum is the most common cause worldwide for superficial dermatophytosis. By their metabolic activity they produce inflammatory responses in the form of erythema, scaling, postulation and micro-abscess formation giving rise to itching and discomfort. The high prevalence rate of superficial dermatophytic infection has shown that 20-25% of the world’s population. The diagnosis was confirmed on the basis of clinical presentation, KOH examination of skin and would light examination. The article also discusses the main treatments for superficial dermatophytosis, with a practical approach to the most commonly used topical and systemic drugs, referring also to their dosage and duration of use in different types of the dermatophytic infection. Keywords: Dermatophytosis; Trichophyton rubrum; Tinea; KOH examination.

Phylogenomics of a new fungal phylum reveals multiple waves of reductive evolution across Holomycota

Compared to multicellular fungi and unicellular yeasts, unicellular fungi with free-living flagellated stages (zoospores) remain poorly known and their phylogenetic position is often unresolved. Recently, rRNA gene phylogenetic analyses of two atypical parasitic fungi with amoeboid zoospores and long kinetosomes, the sanchytrids Amoeboradix gromovi and Sanchytrium tribonematis, showed that they formed a monophyletic group without close affinity with known fungal clades. Here, we sequence single-cell genomes for both species to assess their phylogenetic position and evolution. Phylogenomic analyses using different protein datasets and a comprehensive taxon sampling result in an almost fully-resolved fungal tree, with Chytridiomycota as sister to all other fungi, and sanchytrids forming a well-supported, fast-evolving clade sister to Blastocladiomycota. Comparative genomic analyses across fungi and their allies (Holomycota) reveal an atypically reduced metabolic repertoire for sanchytrids. We infer three main independent flagellum losses from the distribution of over 60 flagellum-specific proteins across Holomycota. Based on sanchytrids’ phylogenetic position and unique traits, we propose the designation of a novel phylum, Sanchytriomycota. In addition, our results indicate that most of the hyphal morphogenesis gene repertoire of multicellular fungi had already evolved in early holomycotan lineages.

Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f.sp. hordei

In barley (Hordeum vulgare), the function of ROPs appears central to polar cell development and the interaction outcome with parasitic fungi but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP with GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant specific ROP nucleotide exchanger domain (PRONE). We performed phylogenetic analyses and annotated barley PRONE GEFs. HvGEF14 is expressed in leaf epidermal tissue and downregulated after inoculation with Blumeria graminis f.sp. hordei. Protein-protein interaction assays indicate that HvGEF14 and the type I barley ROP protein HvRACB can interact in yeast and in planta. Overexpression of HvGEF14 further recruited the ROP-GTP downstream interactor HvRIC171 to the cell periphery and let to interaction of HvRACB with an extended CRIB (Cdc42/Rac Interactive Binding motif) peptide of HvRIC171 in a similar manner as constitutively activated HvRACB. Finally, the over expression of HvGEF14 led to enhanced susceptibility to fungal entry while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells, which is crucial for fungal penetration success.

Hanseniaspora smithiae sp. nov., a Novel Apiculate Yeast Species From Patagonian Forests That Lacks the Typical Genomic Domestication Signatures for Fermentative Environments

During a survey of Nothofagus trees and their parasitic fungi in Andean Patagonia (Argentina), genetically distinct strains of Hanseniaspora were obtained from the sugar-containing stromata of parasitic Cyttaria spp. Phylogenetic analyses based on the single-gene sequences (encoding rRNA and actin) or on conserved, single-copy, orthologous genes from genome sequence assemblies revealed that these strains represent a new species closely related to Hanseniaspora valbyensis. Additionally, delimitation of this novel species was supported by genetic distance calculations using overall genome relatedness indices (OGRI) between the novel taxon and its closest relatives. To better understand the mode of speciation in Hanseniaspora, we examined genes that were retained or lost in the novel species in comparison to its closest relatives. These analyses show that, during diversification, this novel species and its closest relatives, H. valbyensis and Hanseniaspora jakobsenii, lost mitochondrial and other genes involved in the generation of precursor metabolites and energy, which could explain their slower growth and higher ethanol yields under aerobic conditions. Similarly, Hanseniaspora mollemarum lost the ability to sporulate, along with genes that are involved in meiosis and mating. Based on these findings, a formal description of the novel yeast species Hanseniaspora smithiae sp. nov. is proposed, with CRUB 1602H as the holotype.

Development and degradation of a submontane forest in the Beskid Wyspowy Mountains (Polish Western Carpathians) during the Holocene

Mountain regions harbour high biodiversity; however, in numerous areas, they are strongly degraded by human activity. Our study reconstructs the development of the submontane forest belt (400 and 650 m a.s.l.) in the Beskid Wyspowy Mountains (Western Carpathians, Central Europe) affected by climate, humans, fire, and parasitic fungi during the Holocene. This forest belt is considered the most transformed by the human in the Carpathian region. Our multi-proxy study included analyses of pollen, non-pollen palynomorphs (NPPs), plant macrofossils, micro- and macrocharcoal (size fraction >100 µm, analysed in contiguous sampling), geochemical, and sedimentological markers. The results revealed that Picea abies dominated on the fen subjected to study at ca. 8510–5010 cal. BP. Tilia cordata was a substantial component of the submontane forest between ca. 8510 and 2970 cal. BP and it survived a probable Kretzschmaria deusta outbreak, as well as a period of increased fire activity (ca. 6000 cal. BP). The final retreat of forests with a substantial contribution of Tilia was induced by the expansion of Abies alba, Fagus sylvatica, and partly Carpinus betulus and was preceded by the period of increased fire activity and erosion. From ca. 900 cal. BP human-induced deforestations and agricultural and pastoral activity increased. The modern presence of woodlands with Pinus sylvestris and Larix decidua, in the submontane zone in the Beskid Wyspowy Mountains, is a result of sub-recent anthropogenic afforestation on overgrazed areas. The example of the Zbludza site reveals that changes related to fire and pathogen infections, if they have low magnitudes and new competitive taxa are absent, may be reversible in a forest composed of fire-intolerant tree taxa as Tilia. Nonetheless, the widespread submontane ecosystem degradation and the introduction of alien species hamper the regeneration of forest vegetation typical of the submontane zone.