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AbstractAs the honey bee and bumble bee may suffer from the same or related microbial pathogens, cross contamination from commercially reared Bombusspp. to honey bees and wild bumble bees and vice versa is a major concern. Honey bee-collected pollen to feed commercially reared Bombusspp. is a potential risk. Nosemaspp. is a fungal pathogen in bees. In this study, we developed new quantitative detection tools based on the detection of RNA using a TaqMan-based RT-qPCR for Nosema ceranaeand Nosema apis, with extraction controls based on the actin gene of honey bees and bumble bees, respectively. These tools were subsequently applied to study the epidemiology of N. ceranae, a main disease in honey bees. We screened gamma radiation and cold treatment sterilisation for their efficacy to kill N. ceranaespores fed in sugar water and in pollen to honey bees and bumble bees, respectively. N. ceranaeinfection in adult bumble bees was checked. Spores passing the inter-alimentary track were found but no infection was observed. N. ceranaespores were fed to honey bees. Their presence and multiplication were demonstrated, showing the spores were both viable and infectious. Our results indicate that N. ceranaefound in honey bees cannot infect commercially reared bumble bees (Bombus terrestris) and, that gamma radiation effectively kills N. ceranae. The highly specific and sensitive molecular assays developed, were exploited to detect N. ceranaein pollen and faeces, which would allow more comprehensive epidemiological studies on this important pathogen.

ABSTRACTSporotrichosis is one of the most frequent subcutaneous fungal infections in humans and animals caused by members of the plant-associated, dimorphic genus Sporothrix. Three of the four medically important Sporothrixspecies found in Brazil have been considered asexual as no sexual stage has ever been reported in Sporothrix schenckii, Sporothrix brasiliensis, or Sporothrix globosa. We have identified the mating type (MAT) loci in the S. schenckii(strain 1099-18/ATCC MYA-4821) and S. brasiliensis(strain 5110/ATCC MYA-4823) genomes by using comparative genomic approaches to determine the mating type ratio in these pathogen populations. Our analysis revealed the presence of a MAT1-1locus in S. schenckiiwhile a MAT1-2locus was found in S. brasiliensisrepresenting genomic synteny to other Sordariomycetes. Furthermore, the components of the mitogen-activated protein kinase (MAPK)-pheromone pathway, pheromone processing enzymes, and meiotic regulators have also been identified in the two pathogens, suggesting the potential for sexual reproduction. The ratio of MAT1-1to MAT1-2was not significantly different from 1:1 for all three Sporothrixspecies, but the population of S. brasiliensisin the outbreaks originated from a single mating type. We also explored the population genetic structure of these pathogens using sequence data of two loci to improve our knowledge of the pattern of geographic distribution, genetic variation, and virulence phenotypes. Population genetics data showed significant population differentiation and clonality with a low level of haplotype diversity in S. brasiliensisisolates from different regions of sporotrichosis outbreaks in Brazil. In contrast, S. schenckiiisolates demonstrated a high degree of genetic variability without significant geographic differentiation, indicating the presence of recombination. This study demonstrated that two species causing the same disease have contrasting reproductive strategies and genetic variability patterns.

ABSTRACTA new species of nonsporulating fungus, isolated in a case of black-grain mycetoma in Sudan, is described as Madurella fahalii. The species is characterized by phenotypic and molecular criteria. Multigene phylogenies based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS), the partial ß-tubulin gene (BT2), and the RNA polymerase II subunit 2 gene (RPB2) indicate that M. fahaliiis closely related to Madurella mycetomatisand M. pseudomycetomatis; the latter name is validated according to the rules of botanical nomenclature. Madurella ikedaewas found to be synonymous with M. mycetomatis. An isolate from Indonesia was found to be different from all known species based on multilocus analysis and is described as Madurella tropicana. Madurellais nested within the order Sordariales, with Chaetomiumas its nearest neighbor. Madurella fahaliihas a relatively low optimum growth temperature (30°C) and is less susceptible to the azoles than other Madurellaspecies, with voriconazole and posaconazole MICs of 1 µg/ml, a ketoconazole MIC of 2 µg/ml, and an itraconazole MIC of >16 µg/ml. Since eumycetoma is still treated only with azoles, correct species identification is important for the optimal choice of antifungal therapy.

A new species of nonsporulating fungus, isolated in a case of black-grain mycetoma in Sudan, is described as Madurella fahalii. The species is characterized by phenotypic and molecular criteria. Multigene phylogenies based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS), the partial β-tubulin gene (BT2), and the RNA polymerase II subunit 2 gene (RPB2) indicate that M. fahalii is closely related to Madurella mycetomatis and M. pseudomycetomatis; the latter name is validated according to the rules of botanical nomenclature. Madurella ikedae was found to be synonymous with M. mycetomatis. An isolate from Indonesia was found to be different from all known species based on multilocus analysis and is described as Madurella tropicana. Madurella is nested within the order Sordariales, with Chaetomium as its nearest neighbor. Madurella fahalii has a relatively low optimum growth temperature (30°C) and is less susceptible to the azoles than other Madurella species, with voriconazole and posaconazole MICs of 1 μg/ml, a ketoconazole MIC of 2 μg/ml, and an itraconazole MIC of >16 μg/ml. Since eumycetoma is still treated only with azoles, correct species identification is important for the optimal choice of antifungal therapy.

A healthy soil is often defined as a stable soil system with high levels of biological diversity and activity, internal nutrient cycling, and resilience to disturbance. This implies that microbial fluctuations after a disturbance would dampen more quickly in a healthy than in a chronically damaged and biologically impoverished soil. Soil could be disturbed by various processes, for example addition of a nutrient source, tillage, or drying-rewetting. As a result of any disturbance, the numbers of heterotrophic bacteria and of individual species start to oscillate, both in time and space. The oscillations appear as moving waves along the path of a moving nutrient source such as a root tip. The phase and period for different trophic groups and species of bacteria may be shifted indicating that succession occurs. DGGE, Biolog and FAME analysis of subsequent populations in oscillation have confirmed that there is a cyclic succession in microbial communities. Microbial diversity oscillates in opposite direction from oscillations in microbial populations. In a healthy soil, the amplitudes of these oscillations will be small, but the background levels of microbial diversity and activity are high, so that soil-borne diseases will face more competitors and antagonists. However, soil-borne pathogens and antagonists alike will fluctuate in time and space as a result of growing plant roots and other disturbances, and the periods and phases of the oscillations may vary. As a consequence, biological control by members of a single trophic group or species may never be complete, as pathogens will encounter varying populations of the biocontrol agent on the root surface. A mixture of different trophic groups may provide more complete biological control because peaks of different trophic groups occur at subsequent locations along a root. Alternatively, regular addition of soil organic matter may increase background levels of microbial activity, increase nutrient cycling, lower the concentrations of easily available nutrient sources, increase microbial diversity, and enhance natural disease suppression.

Intra- and interspecies transfer of dsRNA viruses between blackAspergilliandAspergillus nidulansstrains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of blackAspergillusandA. nidulansstrains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found amongA. nidulansstrains, transfer among blackAspergilliwas frequently unsuccessful. The lack of virus transfer between blackAspergillusisolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible blackAspergilli. The inefficient transfer of dsRNA viruses between blackAspergilliis not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of blackAspergilliare discussed.

Abstract: The extent of heterokaryon (also termed somatic or vegetative) incompatibility among black Aspergillus strains was examined using nitrate non-utilising mutants selected on chlorate medium. Pairings of complementary mutants showed that somatic compatibility between different strains is exceptional in natural populations of the asexual black Aspergilli. Mycoviruses are present in a considerable fraction of the sampled natural population, but surprisingly, horizontal transfer of mycoviruses only occurs – at least under laboratory conditions – between the (very rare) compatible combinations of strains. Thus, unlike other fungal species, somatic incompatibility in black Aspergilli efficiently blocks virus transfer. Viruses present in black Aspergillus isolates are highly efficiently transmitted to asexual progeny.