(R)-(+)-Lasiodiplodin isolated from the endophytic fungus Sordaria tamaensis exhibits potent antimycobacterial and anti-inflammatory activities in vitro and in vivo: a dual approach for the treatment of severe pulmonary tuberculosis.

OBJECTIVES: This study aimed to evaluate endophytic fungi isolated from Tocoyena bullata and Humiria balsamifera plant species for their antimycobacterial and anti-inflammatory activities, focusing on severe pulmonary tuberculosis cases which are often associated with exacerbated inflammation. METHODS: Mycobacterium suspensions were incubated with the samples for 5 days. RAW 264.7 macrophages stimulated with LPS were also incubated with them for 24 h to assess the inhibition of inflammatory mediator production and cytotoxicity. C57BL/6 mice were infected with Mtb M299 and treated for 15 days with lasiodiplodin (Lasio). KEY FINDINGS: Endophytic fungus Sordaria tamaensis, obtained from T. bullata, was the most promising. Its ethanolic extract impaired mycobacterial growth with MIC50 (µg/ml): 1.5 ± 0.6 (BCG), 66.8 ± 0.1 (H37Rv) and 80.0 ± 0.1 (M299). (R)-(+)-Lasio showed MIC50 92.2 ± 1.8 µg/ml (M299). In addition, Lasio was able to inhibit NO, IL-1ß and TNF-α production and was not cytotoxic for macrophages. M. tuberculosis-infected C57BL/6 animals treated by Lasio reduced the number of acid-fast bacilli, lung pathology, leucocyte influx and proinflammatory cytokine production in the lungs. The class IIa fructose 1,6-bisphosphate aldolase was the predicted hypothetical target of Lasio. CONCLUSIONS: (R)-(+)-Lasio stood out as a promising anti-TB compound, exhibiting anti-inflammatory and antimycobacterial effects, as well as low cytotoxicity.

Resin cast impressions as a tool for microscopic observations of fungal epiphytes on leaves.

A simple method for fungal epiphyte microscopic observations and preservation is described. A two-part clear casting resin, cotton leaves and two species of fungi were used to validate this protocol. We obtained very detailed images of fungal structures using this approach in addition to retaining the impressions for future reference.

Identification and molecular characterization of Subramaniula asteroides causing human fungal keratitis: a case report.

BACKGROUND: Keratitis due to by filamentous fungi are not easy to diagnose thus causing a delay in correct therapy. There are many descriptions of keratitis due to Candida, Fusarium and Aspergillus genera. Subramaniula genus has only recently been reported to cause human infections and there are few descriptions of eye infections due to this filamentous fungus. Diagnosis of fungal keratitis is usually based on microscopic and cultural techniques of samples obtained by corneal swabbing or scraping. Considering the amount of time required to obtain culture results it is wise to use other diagnostic methods, such as molecular analyses. Therapeutic options against these fungi are limited by low tissue penetration in the eye due to ocular barriers. We describe the first case of S. asteroides human keratitis treated with isavuconazole. CASE PRESENTATION: We describe a rare case of fungal keratitis unresponsive to antimicrobial treatment in a 65-year-old male patient without a history of diabetes or immunological diseases. He reported that the onset of symptoms occurred during a long holiday in Cape Verde Island. Initial treatment with topical antibiotics associated to steroids were ineffective, allowing a slow clinical progression of disease to corneal perforation. On admission in our Hospital, slit-lamp examination of the left eye showed conjunctival congestion and hyperemia, a large inferior corneal ulceration with brown pigment, corneal edema, about 3 mm of hypopyon and irido-lenticular synechiae. The slow clinical progression of the disease to corneal perforation and the aspect of the ulcer were consistent with a mycotic etiology. Molecular methods used on fungal colonies isolated by Sabouraud's dextrose agar cultures allowed the identification of Subramaniula asteroids from corneal scraping. Antimicrobial test showed a good susceptibility of this filamentous fungus to voriconazole and isavuconazole. Moreover, this fungal keratitis was successfully treated with isavuconazole, without side effects, observing a progressive clinical improvement. CONCLUSIONS: Molecular methods may be useful for the identification of filamentous fungal keratitis on scraping samples thus shortening the time of diagnosis. Systemic therapy by isavuconazole could be useful to treat the filamentous fungal keratitis, reducing the possible adverse effects due to the use of voriconazole by systemic administration.

Molecular phylogeny, identification and pathogenicity of Rhizopus oryzae associated with root rot of mulberry in India.

AIMS: Root rot caused by a group of fungi is a serious disease in mulberry. This study aims to identify and characterize Rhizopus oryzae and other fungal species associated with root rot of mulberry in India. METHODS AND RESULTS: Rotted root samples were collected from the mulberry gardens from four states of Southern India. The majority of the isolates identified were R. oryzae, and others were saprophytic fungi, less abundant to occasional. Two methods of inoculations were tested to confirm the pathogenicity of the selected isolates and R. oryzae was found to be pathogenic on susceptible mulberry genotypes RC2 and SRDC-1. Multi gene phylogenetic analyses using the internal transcribed spacer region (ITS), actin (ACT) and translation elongation factor 1-α (TEF), identified the isolates as R. oryzae. Additionally, Ovatospora brasiliensis, Amesia nigricolor, Gongronella butleri, Myrmecridium schulzeri, Scedosporium boydii, Graphium euwallacea, Clonostachys rosea andTalaromyces spp. were also identified. CONCLUSION: This study revealed the existence of eleven species of fungi including the first report of R. oryzae and the occurrence of weak pathogens or saprophytes that are associated with the root rot of mulberry in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of R. oryzae causing Rhizopus rot of mulberry in India. Moreover, the occurrence of saprophytes associated with root rot of mulberry was identified. Further studies should focus more on the ability of these species to generate secondary metabolites and extracellular lytic enzymes as they are beneficial for the management of root rot disease.

Reevaluating Cryphonectriaceae and allied families in Diaporthales.

The Diaporthales (Sordariomycetes) includes many important families of plant pathogenic fungi, such as the notorious Cryphonectriaceae. The aim of the present study was to reevaluate this family, along with other families in Diaporthales. Based on phylogenetic analyses using combined sequence data of the internal transcribed spacer (ITS) region, large subunit of the nrDNA (28S), and the translation elongation factor 1-alpha (tef1-α) and DNA-directed RNA polymerase II second largest subunit (rpb2) genes, Cryphonectriaceae is separated into two subclades, comprising 21 genera and 55 species. Foliocryphiaceae, fam. nov., is morphologically and phylogenetically its closest relative but is distinct due to its phylogeny and dimorphic conidia. Mastigosporellaceae, fam. nov., is distinguished from other families in Diaporthales by owning apical conidial appendages. Neocryphonectria, gen. nov., within the family Foliocryphiaceae, with two species associated with Carpinus canker in China, is characterized by fusoid, aseptate macroconidia. Additionally, two new combinations are proposed, namely, Cryphonectria citrine, based on Chromendothia citrine, and Cytospora viridistroma, based on Endothia viridistoma. Based on results obtained in this study, 31 families are accepted into Diaporthales.

Hahyoungchilella caricis gen. nov., sp. nov., isolated from a rhizosphere mudflat of a halophyte (Carex scabrifolia), transfer of Thioclava arenosa Thongphrom et al. 2017 to Pseudothioclava as Pseudothioclava arenosa gen. nov., comb. nov. and proposal of Thioclava electrotropha Chang et al. 2018 as a later heterosynonym of Thioclava sediminum.

A Gram-stain-negative strictly aerobic, marine bacterium, designated GH2-2T, was isolated from a rhizosphere mudflat of a halophyte (Carex scabrifolia) in Gangwha Island, the Republic of Korea. The cells of the organism were oxidase-positive, catalase-positive, flagellated, short rods that grew at 10-40°C, pH 4-10, and 0-13% (w/v) NaCl. The predominant ubiquinone was Q-10. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. The major fatty acid is C18:1. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel isolate formed an independent lineage at the base of the radiation encompassing members of the genus Thioclava, except for Thioclava arenosa. The closest relatives were T. nitratireducens (96.03% sequence similarity) and T. dalianensis (95.97%). The genome size and DNA G+C content were 3.77 Mbp and 59.6 mol%, respectively. Phylogenomic analysis supported phylogenetic distinctness based on 16S rRNA gene sequences. Average nucleotide identity values were 73.6-74.0% between the novel strain and members of the genus Thioclava. On the basis of data obtained from a polyphasic approach, the strain GH2-2T (= KCTC 62124T = DSM 105743) represents a novel species of a new genus for which the name Hahyoungchilella caricis gen. nov., sp. nov. is proposed. Moreover, the transfer of Thioclava arenosa Thongphrom et al. 2017 to Pseudothioclava gen. nov. as Pseudothioclava arenosa comb. nov. is also proposed. Finally, Thioclava electrotropha Chang et al. 2018 is proposed to be a later heterosynonym of Thioclava sediminum Liu et al. 2017.

New fungal cephalothecoid-like fructifications from central European Neogene deposits.

Fragments of cephalothecoid fructifications (peridia) were encountered during palynological investigations of Neogene deposits in Mizerna-Nowa/Poland and Adendorf/Germany. Isolated plates of cephalothecoid ascoma in shape and cellular structure similar to the extant members of the family Cephalothecaceae are described as Cephalothecoidomyces neogenicus fossil gen. et sp. nov. while remnants of fungal sporocarps with cephalothecoid walls with indistinct lines of dehiscence, similar in structure to peridia with cephalothecoid morphology of extant representatives the family Chaetomiaceae (mainly genus Chaetomidium) are assigned to Adendorfia miocenica fossil gen. et sp. nov. We also propose a new interpretation of some previously described fossil fungal taxa that we consider to be remnants of cephalothecoid ascomata.

Microbiota Characterization of Compost Using Omics Approaches Opens New Perspectives for Phytophthora Root Rot Control.

Phytophthora root rot caused by Phytophthora nicotianae is an economically important disease in pepper crops. The use of suppressive composts is a low environmental impact method for its control. Although attempts have been made to reveal the relationship between microbiota and compost suppressiveness, little is known about the microorganisms associated with disease suppression. Here, an Ion Torrent platform was used to assess the microbial composition of composts made of different agro-industrial waste and with different levels of suppressiveness against P. nicotianae. Both bacterial and fungal populations responded differently depending on the chemical heterogeneity of materials used during the composting process. High proportions (67-75%) of vineyard pruning waste were used in the most suppressive composts, COM-A and COM-B. This material may have promoted the presence of higher relative abundance of Ascomycota as well as higher microbial activity, which have proved to be essential for controlling the disease. Although no unique fungi or bacteria have been detected in neither suppressive nor conducive composts, relatively high abundance of Fusarium and Zopfiella were found in compost COM-B and COM-A, respectively. To the best of our knowledge, this is the first work that studies compost metabolome. Surprisingly, composts and peat clustered together in principal component analysis of the metabolic data according to their levels of suppressiveness achieved. This study demonstrated the need for combining the information provided by different techniques, including metagenomics and metametabolomics, to better understand the ability of compost to control plant diseases.

Cephalotheca sulfurea (Ascomycota, Sordariomycetes), a new fungal pathogen of the farmed rainbow trout Oncorhynchus mykiss.

The first case of visceral mycotic infection due to Cephalotheca sulfurea (Cephalothecaceae, Ascomycota) is documented in farmed rainbow trout from a raceway culture system. The disease clinically manifested as a hyperaemic area in the liver of the fish, and histological examination using silver and PAS staining showed the presence of numerous foci of hyphae and spores. The causative agent was first isolated in pure culture from the liver and identified using morphological characteristics. Sequence data from ITS and LSU rDNA also clearly confirmed C. sulfurea as the causal agent. The pathogenicity of related species belonging to the family Cephalothecaceae has been well-documented in humans and dogs (superficial as well as systemic infections). However, C. sulfurea has never been reported as a pathogen of humans or animals, including marine and freshwater fishes. The morphological identification of C. sulfurea is difficult due to its similarity to several different fungal genera, and molecular methods are strongly recommended for reliable identification.

Revisiting the Life Cycle of Dung Fungi, Including Sordaria fimicola.

Dung fungi, such as Sordaria fimicola, generally reproduce sexually with ascospores discharged from mammalian dung after passage through herbivores. Their life cycle is thought to be obligate to dung, and thus their ascospores in Quaternary sediments have been interpreted as evidence of past mammalian herbivore activity. Reports of dung fungi as endophytes would seem to challenge the view that they are obligate to dung. However, endophyte status is controversial because surface-sterilization protocols could fail to kill dung fungus ascospores stuck to the plant surface. Thus, we first tested the ability of representative isolates of three common genera of dung fungi to affect plant growth and fecundity given that significant effects on plant fitness could not result from ascospores merely stuck to the plant surface. Isolates of S. fimicola, Preussia sp., and Sporormiella sp. reduced growth and fecundity of two of three populations of Bromus tectorum, the host from which they had been isolated. In further work with S. fimicola we showed that inoculations of roots of B. tectorum led to some colonization of aboveground tissues. The same isolate of S. fimicola reproduced sexually on inoculated host plant tissues as well as in dung after passage through sheep, thus demonstrating a facultative rather than an obligate life cycle. Finally, plants inoculated with S. fimicola were not preferred by sheep; preference had been expected if the fungus were obligate to dung. Overall, these findings make us question the assumption that these fungi are obligate to dung.

Humicola sp. as a Cause of Peritoneal Dialysis-Associated Peritonitis.

Peritoneal dialysis is the renal replacement modality used by ∼20% of patients with end-stage kidney disease (S. McDonald, P. Clayton, and K. Hurst, p. 6.2-6.27, in ANZDATA 2012 Annual Report, 35th ed., 2012). A major complication of peritoneal dialysis is the development of peritonitis. We describe a case of Humicola sp. causing peritoneal dialysis (PD)-associated peritonitis, successfully treated with a prolonged course of antifungal therapy.

A re-evaluation of the genus Myceliophthora (Sordariales, Ascomycota): its segregation into four genera and description of Corynascus fumimontanus sp. nov.

Based on a number of isolates of Myceliophthora (Chaetomiaceae, Sordariales, Ascomycota) recently isolated from soil samples collected in USA, the taxonomy of the genus was re-evaluated through phylogenetic analyses of sequences from the nuc rDNA internal transcribed spacer region and genes for the second largest subunit of RNA polymerase II and translation elongation factor 1α. Members of Myceliophthora were split into four monophyletic clades strongly supported by molecular and phenotypic data. Such clades correspond with Myceliophthora, now restricted only to the type species of the genus Corynascus, which is re-established with five species, the new monotypic genus Crassicarpon and also the new genus Thermothelomyces (comprising four species). Myceliophthora lutea is mesophilic and a permanently asexual morph compared to the members of the other three mentioned genera, which also are able to sexually reproduce morphs with experimentally proven links to their asexual morphs. The asexual morph of M. lutea is characterized by broadly ellipsoidal, smooth-walled conidia with a wide, truncate base. Crassicarpon thermophilum is thermophilic and heterothallic and produces spherical to cuneiform, smooth-walled conidia and cleistothecial ascomata of smooth-walled, angular cells and ascospores with a germ pore at each end. Corynascus spp. are homothallic and mesophilic and produce spherical, mostly ornamented conidia and cleistothecial ascomata with textura epidermoidea composed of ornamented wall cells, and ascospores with one germ pore at each end. Thermothelomyces spp. are thermophilic, heterothallic and characterized by similar ascomata and conidia as Corynascus spp., but its ascospores exhibit only a single germ pore. A dichotomous key to distinguish Myceliophthora from the other mentioned genera are provided, as well as dichotomous keys to identify the species of Corynascus and Thermothelomyces. A new species, namely Corynascus fumimontanus, characterized by verrucose ascomatal wall cells and irregularly shaped ascospores, is described and illustrated.

The anamorphic genus Monotosporella (Ascomycota) from Eocene amber and from modern Agathis resin.

The anamorphic fungal genus Monotosporella (Ascomycota, Sordariomycetes) has been reco-vered from a piece of Early Eocene Indian amber, as well as from the surface of extant resin flows in New Caledonia. The fossil fungus was obtained from the Tarkeshwar Lignite Mine of Gujarat State, western India, and was part of the biota of an early tropical angiosperm rainforest. The amber inclusion represents the second fossil record of Sordariomycetes, as well as the first fossil of its particular order (either Savoryellales or Chaetosphaeriales). The fossil fungus is distinguished from extant representatives by possessing both short conidiophores and small two-septate pyriform conidia, and is described as Monotosporella doerfeltii sp. nov. Inside the amber, the anamorph is attached to its substrate, which is likely the degraded thallus of a cladoniform lichen. The extant New Caledonian species is assigned to Monotosporella setosa. It was found growing on semi-solidified resin flows of Agathis ovata (Araucariaceae), and is the first record of Monotosporella from modern resin substrates.

Thermophilic fungi in an aridland ecosystem.

We report a comprehensive multi-year study of thermophilic fungi at the Sevilleta National Wildlife Refuge in central New Mexico. Recovery of thermophilic fungi from soils showed seasonal fluctuations, with greater abundance correlating with spring and summer precipitation peaks. In addition to grassland soils, we obtained and characterized isolates from grassland and riparian litter, herbivore dung and biological soil crusts. All strains belonged to either the Eurotiales or Sordariales (Chaetomiaceae). No particular substrate or microhabitat associations were detected. Molecular typing of strains revealed substantial phylogenetic diversity, eight ad hoc phylogroups across the two orders were identified and genetic diversity was present within each phylogroup. Growth tests over a range of temperatures showed substantial variation in maximum growth rates among strains and across phylogroups but consistency within phylogroups. Results demonstrated that 45-50 C represents the optimal temperature for growth of most isolates, with a dramatic decline at 60 C. Most strains grew at 60 C, albeit slowly, whereas none grew at 65 C, providing empirical confirmation that 60 C presents an evolutionary threshold for fungal growth. Our results support the hypothesis that fungal thermophily is an adaptation to transient seasonal and diurnal high temperatures, rather than simply an adaptation to specialized high-temperature environments. We note that the diversity observed among strains and the frequently confused taxonomy within these groups highlight the need for comprehensive biosystematic revision of thermophilic taxa in both orders.

Protein enrichment, cellulase production and in vitro digestion improvement of pangolagrass with solid state fermentation.

BACKGROUND AND PURPOSE: Pangolagrass, Digitaria decumbens Stent, is a major grass for cow feeding, and may be a good substrate for protein enrichment. To improve the quality of pangolagrass for animal feeding, cellulolytic microbes were isolated from various sources and cultivated with solid state fermentation to enhance the protein content, cellulase production and in vitro digestion. The microbes, culture conditions and culture media were studied. METHODS: Cellulolytic microbes were isolated from pangolagrass and its extracts, and composts. Pangolagrass supplemented with nitrogen and minerals was used to cultivate the cellulolytic microbes with solid state fermentation. The optimal conditions for protein enrichment and cellulase activity were pangolagrass substrate at initial moisture 65-70%, initial pH 6.0-8.0, supplementation with 2.5% (NH(4))(2)SO(4), 2.5% KH(2)PO(4) and K(2)HPO(4) mixture (2:1, w/w) and 0.3% MgSO(4).7H(2)O and cultivated at 30(o)C for 6 days. RESULTS: The protein content of fermented pangolagrass increased from 5.97-6.28% to 7.09-16.96% and the in vitro digestion improved from 4.11-4.38% to 6.08-19.89% with the inoculation of cellulolytic microbes by solid state fermentation. Each 1 g of dried substrate yielded Avicelase 0.93-3.76 U, carboxymethylcellulase 1.39-4.98 U and ß-glucosidase 1.20-6.01 U. The isolate Myceliophthora lutea CL3 was the strain found to be the best at improving the quality of pangolagrass for animal feeding with solid state fermentation. CONCLUSION: Solid state fermentation of pangolagrass inoculated with appropriate microbes is a feasible process to enrich protein content, increase in vitro digestibility and improve the quality for animal feeding.

Severe pigmented keratitis caused by Cladorrhinum bulbillosum.

We report a case of severe pigmented keratitis with poor prognosis, caused by Cladorrhinum bulbillosum. Antifungal treatment with topical natamycin and fluconazole eye drops and oral tablet fluconazole failed to heal the ulcer and resulted in perforation. The causative fungus, C. bulbillosum, was identified on the basis of its typical microscopic features and 98% sequence homology to ex-type isolate CBS 304.90 (accession no. FM955448). The results of an in vitro antifungal susceptibility test indicated that the isolate was susceptible to natamycin, amphotericin B, fluconazole and itraconazole. The present case is the third case of keratitis and the second case of human keratitis. Compromised immunity due to liver cirrhosis could lead to a failed prognosis even when the fungal isolate is highly susceptible to antifungal treatment.

Invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient: a new cause of cross-reactivity with the Aspergillus galactomannan serum antigen assay.

Myceliophthora thermophila is a thermophilic mould widely found in the environment but rarely responsible for human infections. We describe a case of invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient with haematological malignancy. Cross-reactivity with Aspergillus galactomannan assay (GM) was demonstrated by repeated positive results and confirmed by cross-reaction between the fungal isolate and the GM assay. The patient was successfully treated with voriconazole. Potential GM cross-reactivity must be considered in future studies including patients categorized as having probable invasive aspergillosis using the GM as the only mycological criterion.

Fatal cerebral phaeohyphomycosis in an immunocompetent individual due to Thielavia subthermophila.

We report the first case of fatal brain infection in an Indian farmer caused by Thielavia subthermophila, a dematiaceous thermophilic fungus in the order Sordariales, and present a review of previous infections from this order. The patient failed amphotericin B therapy combined with surgical excision despite the drug's low MICs in vitro.

Effect of dsRNA on growth rate and reproductive potential of Monosporascus cannonballus.

The effect of double stranded RNA (dsRNA) infection on growth rate and the reproductive potential of Monosporascus cannonballus was studied in 21 isolates collected in cucurbit growing areas of Spain and Tunisia. The isolates were incubated on potato dextrose agar (PDA) under different conditions of temperature, pH, and water potential (Ψ(s)). They showed optimal growth temperatures over the range of 27-34°C and perithecia formation was obtained mainly at 25 and 30°C, although some isolates were able to produce perithecia at 35°C. All isolates were able to produce perithecia in a broad range of pHs (4-8). Regarding the effect of Ψ(s,) the isolates were more tolerant to grow on KCl than on NaCl. For each solute, radial growth decreased progressively as Ψ(s) decreased and was severely limited at -5.0 to -6.0MPa. Perithecia formation was highest at -0.5MPa, decreased at -1.0MPa and occurred just in some isolates at -2.0MPa. Nine of the M. cannonballus isolates harboured dsRNA with 2-6 bands each and a size range of 1.9-18.0Kb. Phenotypical data were subjected to multivariate factorial analysis. Most of the isolates clustered in two groups corresponding with the presence/absence of dsRNA elements. Isolates without detectable dsRNA produced more perithecia. However, isolates with dsRNA produced lower number of perithecia depending on the pH, Ψ(s,) or solute used. These results improve our understanding of the behaviour and growth of this pathogen in soil, and can be useful to implement effective disease control.