Discovery of highly reactive self-splicing group II introns within the mitochondrial genomes of human pathogenic fungi.

Fungal pathogens represent an expanding global health threat for which treatment options are limited. Self-splicing group II introns have emerged as promising drug targets, but their development has been limited by a lack of information on their distribution and architecture in pathogenic fungi. To meet this challenge, we developed a bioinformatic workflow for scanning sequence data to identify unique RNA structural signatures within group II introns. Using this approach, we discovered a set of ubiquitous introns within thermally dimorphic fungi (genera of Blastomyces, Coccidioides and Histoplasma). These introns are the most biochemically reactive group II introns ever reported, and they self-splice rapidly under near-physiological conditions without protein cofactors. Moreover, we demonstrated the small molecule targetability of these introns by showing that they can be inhibited by the FDA-approved drug mitoxantrone in vitro. Taken together, our results highlight the utility of structure-based informatic searches for identifying riboregulatory elements in pathogens, revealing a striking diversity of reactive self-splicing introns with great promise as antifungal drug targets.

Diversity of fungal pathogens associated with loquat and development of novel virulence scales.

Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an important fruit crop in Pakistan; however, a constant decline in its production is noted due biotic and abiotic stresses, particularly disease infestation. Fungal pathogens are the major disease-causing agents; therefore, their identification is necessary for devising management options. This study explored Taxila, Wah-Cantt, Tret, Chatar, Murree, Kalar-Kahar, Choa-Saidan-Shah and Khan-Pur districts in the Punjab and Khyber Paktoon Khawa (KPK) provinces of Pakistan to explore the diversity of fungal pathogens associated with loquat. The samples were collected from these districts and their microscopic characterizations were accomplished for reliable identification. Alternaria alternata, Curvularia lunata, Lasiodiplodia theobromae, Aspergilus flavis, Botrytis cinerea, Chaetomium globosum, Pestalotiopsis mangiferae and Phomopsis sp. were the fungal pathogens infesting loquat in the study area. The isolates of A. alternata and C. lunata were isolated from leaf spots and fruit rot, while the isolates of L. theobromae were associated with twig dieback. The remaining pathogens were allied with fruit rot. The nucleotide evidence of internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) were computed from all the pathogens and submitted in the database of National Center for Biotechnology Information (NCBI). For multigene analysis, beta-tubulin (BT) gene and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were explored for A. alternata and C. lunata isolates, respectively. The virulence scales of leaf spots, fruit rot, and twig dieback diseases of loquat were developed for the first time through this study. It is the first comprehensive study with morpho-molecular identification, and newly developed virulence scales of the fungal pathogens associated with loquat, which improves the understanding of these destructive diseases.

miRNA Mediated Regulation and Interaction between Plants and Pathogens.

Plants have evolved diverse molecular mechanisms that enable them to respond to a wide range of pathogens. It has become clear that microRNAs, a class of short single-stranded RNA molecules that regulate gene expression at the transcriptional or post-translational level, play a crucial role in coordinating plant-pathogen interactions. Specifically, miRNAs have been shown to be involved in the regulation of phytohormone signals, reactive oxygen species, and NBS-LRR gene expression, thereby modulating the arms race between hosts and pathogens. Adding another level of complexity, it has recently been shown that specific lncRNAs (ceRNAs) can act as decoys that interact with and modulate the activity of miRNAs. Here we review recent findings regarding the roles of miRNA in plant defense, with a focus on the regulatory modes of miRNAs and their possible applications in breeding pathogen-resistance plants including crops and trees. Special emphasis is placed on discussing the role of miRNA in the arms race between hosts and pathogens, and the interaction between disease-related miRNAs and lncRNAs.

Searching for New Beneficial Bacterial Isolates of Wild Raspberries for Biocontrol of Phytopathogens-Antagonistic Properties and Functional Characterization.

The threat caused by plants fungal and fungal-like pathogens is a serious problem in the organic farming of soft fruits. The European Commission regulations prohibit some commercially available chemical plant protection products, and instead recommend the use of natural methods for improving the microbial soil status and thus increasing resistance to biotic stresses caused by phytopathogens. The solution to this problem may be biopreparations based on, e.g., bacteria, especially those isolated from native local environments. To select proper bacterial candidates for biopreparation, research was provided to preliminarily ensure that those isolates are able not only to inhibit the growth of pathogens, but also to be metabolically effective. In the presented research sixty-five isolates were acquired and identified. Potentially pathogenic isolates were excluded from further research, and beneficial bacterial isolates were tested against the following plant pathogens: Botrytis spp., Colletotrichum spp., Phytophthora spp., and Verticillium spp. The eight most effective antagonists belonging to Arthrobacter, Bacillus, Pseudomonas, and Rhodococcus genera were subjected to metabolic and enzymatic analyses and a resistance to chemical stress survey, indicating to their potential as components of biopreparations for agroecology.

Hongos anamorfos biodegradan los libros del Área Histórica de la Universidad Central del Ecuador (UCE) / Anamorphous fungi biodegrade the books of the Historical Area from the Central University of Ecuador (CUE)

Determinamos los géneros de hongos anamorfos que contaminan los libros del área de cuarentena y limpieza, dentro del Área Histórica de la Universidad Central del Ecuador (UCE). Realizamos un hisopado aleatorio a una muestra representativa de 50 de estos libros de acuerdo a una Tabla militarizada estándar. También hisopamos como muestra preferencial a 21 libros gravemente contaminados con hongos. Los hisopados tuvieron una superficie de 5x5 cm, friccionando en la pasta, el borde y el interior de estos libros. Las 213 muestras tomadas fueron inoculadas en medio de cultivo Agar Malta. Los medios fueron incubados a una temperatura de 28°C durante 7 días. Realizamos observaciones por microscopía a 40 y 100x además de usar literatura especializada para la identificación hasta el nivel de género de hongos anamorfos. Los géneros más abundantes en este estudio fueron Penicillium (80,2%) y Mucor (8,1%). (AU)

We determined the genera of anamorphic fungi that contaminate the books in the quarantine and cleaning area, within the Historical Area of the Central University of Ecuador (CUE). We performed a random swab on a representative sample of 50 of these books according to a standard militarized Table. We also swabbed as a preferential sample 21 books seriously contaminated with fungi. The swabs had a surface area of 5x5 cm, rubbing on the paste, the edge and the interior of these books. The 213 samples taken were inoculated in Agar Malta culture medium. The media were incubated at a temperature of 28° C for 7 days. We made observations by microscopy at 40 and 100x in addition to using specialized literature for the identification down to the genus level of anamorphic fungi. The most abundant genus in this study were Penicillium(80,2%) and Mucor(8,1%). (AU)

Evaluation of soybean genotypes for reaction to natural field infection by Cercospora species causing purple seed stain.

Purple seed stain (PSS) of soybean (Glycine max (L.) Merr.) is a prevalent seed disease. It results in poor seed quality and reduced seed lot market grade, and thus undermines value of soybean worldwide. The objectives of this research were to evaluate the reaction of selected soybean genotypes collected from 15 countries representing maturity groups (MGs) III, IV, and V to PSS, and to identify new sources of resistance to PSS based on three years of evaluation of natural field infection by Cercospora spp. in the Mississippi Delta of the U. S. In this study, 42 soybean genotypes were evaluated in 2010, 2011, and 2012. Seventeen lines including six MG III (PI 88490, PI 504488, PI 417361, PI 548298, PI 437482, and PI 578486), seven MG IV (PI 404173, PI 346308, PI 355070, PI 416779, PI 80479, PI 346307, and PI 264555), and four MG V (PI 417567, PI 417420, PI 381659, and PI 407749) genotypes had significantly lower percent seed infection by Cercospora spp. than the susceptible checks and other genotypes evaluated (P ≤ 0.05). These genotypes of soybean can be used in developing soybean cultivars or germplasm lines with resistance to PSS and for genetic mapping of PSS resistance genes. In addition, among these 17 lines with different levels of resistance to PSS, nine soybean genotypes (PI 417361, PI 504488, PI 88490, PI 346308, PI 416779, PI 417567, PI 381659, PI 417567, and PI 407749) were previously reported as resistant to Phomopsis seed decay. Therefore, they could be useful in breeding programs to develop soybean cultivars with improved resistance to both seed diseases.

Reactive Oxygen Species Localization Programs Inflammation to Clear Microbes of Different Size.

How the number of immune cells recruited to sites of infection is determined and adjusted to differences in the cellular stoichiometry between host and pathogen is unknown. Here, we have uncovered a role for reactive oxygen species (ROS) as sensors of microbe size. By sensing the differential localization of ROS generated in response to microbes of different size, neutrophils tuned their interleukin (IL)-1ß expression via the selective oxidation of NF-κB, in order to implement distinct inflammatory programs. Small microbes triggered ROS intracellularly, suppressing IL-1ß expression to limit neutrophil recruitment as each phagocyte eliminated numerous pathogens. In contrast, large microbes triggered ROS extracellularly, amplifying IL-1ß expression to recruit numerous neutrophils forming cooperative clusters. Defects in ROS-mediated microbe size sensing resulted in large neutrophil infiltrates and clusters in response to small microbes that contribute to inflammatory disease. These findings highlight the impact of ROS localization on signal transduction.

Codon optimization underpins generalist parasitism in fungi.

The range of hosts that parasites can infect is a key determinant of the emergence and spread of disease. Yet, the impact of host range variation on the evolution of parasite genomes remains unknown. Here, we show that codon optimization underlies genome adaptation in broad host range parasites. We found that the longer proteins encoded by broad host range fungi likely increase natural selection on codon optimization in these species. Accordingly, codon optimization correlates with host range across the fungal kingdom. At the species level, biased patterns of synonymous substitutions underpin increased codon optimization in a generalist but not a specialist fungal pathogen. Virulence genes were consistently enriched in highly codon-optimized genes of generalist but not specialist species. We conclude that codon optimization is related to the capacity of parasites to colonize multiple hosts. Our results link genome evolution and translational regulation to the long-term persistence of generalist parasitism.

Especies oportunistas de importancia clínica de los géneros bipolaris shoemaker y curvularia boedijn: su caracterización bajo los nuevos criterios taxonómicos / Opportunistic species of clinical relevance in the bipolaris shoemaker & curvularia boedijn genera: its characterization under newest taxonomic criteria

Los géneros fúngicos Bipolaris y Curvularia,incluyen numerosas especies fitopatógenas,saprotrofas y algunas oportunistas emergentes enel ser humano como en otros animales. La distribuciónecológica de ambos taxas es cosmopolita enla gran mayoría de sus integrantes, encontrándosefrecuentemente en hojas, tallos y raíces de diferentespastos, en especial Poaceae, pero tambiénen una gran variedad de dicotiledoneas, aire y sueloen todos los continentes. Cochliobolus, Bipolarisy Curvularia integran un complejo de especiestaxonómicamente confuso, debido a los constantescambios en la nomenclatura de algunos de sus integrantesasexuales (Bipolaris y Curvularia), loscuales se han diferenciado principalmente en basea la morfología de sus conidios, situación a vecesmuy dificultosa debido a que en ambos génerosalgunas especies presentan similares característicasconidiales. Una cuidadosa identificación y unanominación precisa de las especies es crucial parael acceso a la información referente en la literaturamoderna, que gracias a la biología molecularha resuelto muchas de las dudas de la taxonomíabasada solo en el fenotipo.Las especies de ambos géneros (principalmenteCurvularia) pueden afectar a pacientesinmunocomprometidos e inmunocompetentes,en especial B. cynodontis, C. australiensis C.hawaiensis y C. spicifera, causando cuadros clínicosdiversos ya sea superficiales o profundos enmuchas áreas geográficas...

The fungal genera Bipolaris and Curvularia,include numerous plant pathogenic species,saprophitic and some emerging opportunistic inman and other animals. The ecological distributionof both taxa is cosmopolitan in the vast majority ofits members, often being in leaves, stems and rootsof different grasses, especially Poaceae, but also ina variety of dicotyledonous, air and soil on all continents. Cochliobolus, Bipolaris and Curvulariaintegrate a complex of species taxonomically confusingdue to the constant changes in the nomenclatureof some of its asexual members (Bipolarisand Curvularia), which are differentiated mainlybased on the morphology of its conidia, a situationsometimes very difficult because in both generaconidial of some species have similar characteristics. Careful identification and a precise nominationof species is crucial for access to informationconcerning in modern literature, actually thanks tomolecular biology has solved many of the doubtsof taxonomy based only on the phenotype.The species of both genera (mainly Curvularia)can affect immunocompetent or debilitatepatients, especially B. cynodontis, C. australiensis,C. hawaiensis and C.spicifera, causing varioussuperficial or systemic clinical cases in manygeographic areas...

Invasive cutaneous Neoscytalidium infections in renal transplant recipients: a series of five cases.

BACKGROUND: Neoscytalidium species (formerly Scytalidium species) are black fungi that usually cause cutaneous infections mimicking dermatophytes lesions. Very few publications have reported invasive or disseminated infections. CASE PRESENTATION: In this paper, we report the clinical presentations, treatments and outcomes of five cases of invasive Neoscytalidium infections with cutaneous involvement, including two cases with disseminated infection, in five renal transplant recipients. To our knowledge, this is the first report of a series-albeit small-of renal transplant patients in whom this infection was identified. All cases occurred in a single hospital in Paris, France, between 2001 and 2011. Patients all originate from tropical area. CONCLUSION: Treatments of Neoscytalidium infection varied greatly, underlining the lack of a recommendation for a standardized treatment. All patients were cured after long-term antifungal therapy and/or surgical excision. Interestingly, one patient with disseminated infection involving the left elbow, the right leg, the lungs and the nasal septum was cured by medical therapy only without surgery. This may suggest that in contrast to others mycoses (such as mucormycosis), an adequate medical treatment could be sufficient for treating Neoscytalidium. We also point out the difficulties we had in diagnosing two patients with Kaposi's sarcoma because of the similarity of the lesions. Furthermore, our report underlines the need to check for this rare infection in immunocompromised kidney transplant recipients originating from tropical areas.

Pathogenicity of conidia-based preparations of entomopathogenic fungi against the greenhouse pest aphids Myzus persicae, Aphis gossypii, and Aulacorthum solani (Hemiptera: Aphididae).

Seeking new isolates of entomopathogenic fungi with greater virulence against greenhouse aphid pests than those currently registered in North America for control of these insects, single-dose screening assays of 44 selected fungal isolates and 4 commercially available strains were conducted against first-instar nymphs of Myzus persicae and Aphis gossypii. The assays identified a number of Beauveria and Metarhizium isolates with virulence equal to or greater than that of the commercial strains against the nymphal aphids, but none exhibited exceptionally high virulence. Virulence of Isaria isolates was unexpectedly low (<31% mortality at doses>1000conidia/mm(2)). In dose-response assays, Beauveria ARSEF 5493 proved most virulent against M. persicae and A. gossypii; however, LC50s of this isolate did not differ significantly from those of B. bassiana commercial strain JW-1. Dose-response assays were also conducted with Aulacorthum solani, the first reported evaluations of Beauveria and Metarhizium against this pest. The novel isolate Metarhizium 5471 showed virulence⩾that of Beauveria 5493 in terms of LC25 and LC50, but 5493 produced a steeper dose response (slope). Additional tests showed that adult aphids are more susceptible than nymphs to fungal infection but confirmed that infection has a limited pre-mortem effect on aphid reproduction. Effects of assay techniques and the potential of fungal pathogens as aphid-control agents are discussed.

[Formation peculiarities of Quercus robur L. acorn mycobiota under conditions of Kyiv Polissya].

The structure of mycobiota (epiphytic and from inner tissues) of Q. robur acorns (580 samples) under conditions of Kyiv Polissya was studied. Acorn samples were collected from oak trees during vegetation period. The epiphytes and mycobiota isolated from inner tissues of acorns was represented by 38 and 20 species, respectively. The majority of fungi were isolated in July. Cosmopolitan and non-specific species Acremonium sp., Alternaria alternata, Cladosporium cladosporioides, Trichoderma harzianum, T. viride and specific ones - Phoma sp. and Phomopsis sp. were isolated from inner acorn tissues. It was established that potential seed and seedling pathogens infect acorns during the process of their formation. The paper is presented in Ukrainian.

Complexes of potentially pathogenic microscopic fungi in anthropogenic polluted soils.

This study was undertaken to investigate the species' diversity and structure of potentially pathogenic microscopic fungal complexes in podzolic soils polluted by fluorine, heavy metals (Cu, Ni, Co), oil products (diesel fuel, gas condensate, mazut). Lists of potentially pathogenic fungi isolated from soils are made specifically for north-western part of Russia (Kola Peninsula). The majority of studied fungus species belong to the following genera: Penicillium, Aspergillus, Mucor, Lecanicillium and Phoma. Penicillium miczynskii was identified as the most stable type of fungus with respect to all studied types of oil products. Mucor hiemalis was identified as the most sensitive type. An increase of 15% portion of potentially pathogenic fungi as compared to the background soil in zones of aluminum and copper-nickel plants was revealed. The results indicate an increase of 20-25% of potentially pathogenic fungi in pollution of soil with oil products. The structure of fungal complexes was observed to have changed in the polluted soils and the species number and frequency of occurrence of potentially pathogenic fungi were also increased.

Curvularia brain abscess.

Curvularia is a ubiquitous dematiaceous fungus that is a very rare but often fatal cause of infection in the central nervous system (CNS). In this report, we describe a patient with chronic sinusitis who presented with a Curvularia abscess of the skull base extending into the left frontal lobe. She was successfully treated with aggressive surgical resection and antibiotic therapy. In the published literature, this patient to our knowledge represents the longest period of disease-free follow-up in those afflicted with CNS Curvularia infection, indicating the importance of proper diagnosis and aggressive surgical debridement for a successful outcome.

Eplt4 proteinaceous elicitor produced in Pichia pastoris has a protective effect against Cercosporidium sofinum infections of soybean leaves.

A complementary DNA library was constructed from the mycelium of Trichoderma asperellum T4, and a highly expressed gene fragment named EplT4 was found. In order to find a more efficient and cost-effective way of obtaining EplT4, this study attempted to produce EplT4 using a Pichia pastoris expression system. The gene encoding EplT4, with an additional 6-His tag at the C-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115 to obtaining more protein for the further research. Transformants of P. pastoris were selected by PCR analysis, and the ability to secrete high levels of the EplT4 protein was determined. The optimal conditions for induction were assayed using the shake flask method and an enzyme-linked immunosorbent assay. The yield of purified EplT4 was approximately 20 mg/L by nickel affinity chromatography and gel-filtration chromatography. Western blot and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis revealed that the recombinant EplT4 was expressed in both its monomers and dimers. Soybean leaves treated with the EplT4 monomer demonstrated the induction of glucanase, chitinase III-A, cysteine proteinase inhibitor, and peroxidase genes. Early cellular events in plant defense response were also observed after incubation with EplT4. Soybean leaves protected by EplT4 against the pathogen Cercosporidium sofinum (Hara) indicated that EplT4 produced in P. pastoris was biologically active and would be potentially useful for improving food security.

Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses.

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

Growth rate inhibition of phytopathogenic fungi by characterized chitosans

The inhibitory effects of fifteen chitosans with different degrees of polymerization (DP) and different degrees of acetylation (F A) on the growth rates (GR) of four phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer) were examined using a 96-well microtiter plate and a microplate reader. The minimum inhibitory concentrations (MICs) of the chitosans ranged from 100 µg × mL-1 to 1,000 µg × mL-1 depending on the fungus tested and the DP and F A of the chitosan. The antifungal activity of the chitosans increased with decreasing F A. Chitosans with low F A and high DP showed the highest inhibitory activity against all four fungi. P. expansum and B. cinerea were relatively less susceptible while A. alternata and R. stolonifer were relatively more sensitive to the chitosan polymers. Scanning electron microscopy of fungi grown on culture media amended with chitosan revealed morphological changes.

Perspectives on the potential of entomopathogenic fungi in biological control of ticks.

Ticks are serious health threats for humans, and both domestic and wild animals. Ticks are controlled mostly by application of chemical products; but these acaricides have several negative side effects, including toxicity to animals, environmental contamination, and induction of chemical resistance in some tick populations. Entomopathogenic fungi infect arthropods in nature and can occur at enzootic or epizootic levels in their host populations. Laboratory studies clearly demonstrate that these fungi can cause high mortality in all developmental stages of several tick species, and also reduce oviposition of infected engorged females. Tick mortality following application of fungi in the field, however, often is less than that suggested by laboratory tests. This is due to many negative biotic and climatic factors. To increase efficacy of fungal agents for biological control of ticks under natural conditions, several points need consideration: (1) select effective isolates (viz., high virulence; and tolerance to high temperature, ultraviolet radiation and desiccation); (2) understand the main factors that affect virulence of fungal isolates to their target arthropods including the role of toxic metabolites of the fungal isolates; and (3) define with more precision the immune response of ticks to infection by entomopathogenic fungi. The current study reviews recent literature on biological control of ticks, and comments on the relevance of these results to advancing the development of fungal biocontrol agents, including improving formulation of fungal spores for use in tick control, and using entomopathogenic fungi in integrated pest (tick) management programs.