Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

Phylogeny of the genus Fuscoporia and taxonomic assessment of the F. contigua group.

The genus Fuscoporia of the Hymenochaetaceae is characterized by resupinate to pileate basidiocarps, a dimitic hyphal system with fine crystal aggregates and encrusted generative hyphae in dissepiment edge and tube trama, the presence of hymenial setae, and hyaline, thin-walled, smooth basidiospores. Members of the F. contigua group are easy to distinguish from other species of Fuscoporia because of the moderately large pores, presence of mycelial setae, and large hymenial setae. Here, we explore phylogenetic relationships among 20 species of Fuscoporia based on examination of some 90 collections sampled worldwide. Seven new species are recognized in the F. contigua group-F. americana, F. centroamericana, F. costaricana, F. latispora, F. monticola, F. septiseta, and F. sinica-described from China, Costa Rica, Mexico, and the United States. Phylogenetic analyses inferred from DNA sequences of the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), D1-D2 domains of nuc 28S rDNA (28S), and translation elongation factor EF-1 alpha (tef1) support the F. contigua group as one of two major clades within Fuscoporia comprising nine species worldwide.

[Quantitative Assessment of the Combined Effect of the Nitrogen Status, Light and Dehydration of Mycelium on Conidiation in Neurospora crassa].

We performed a quantitative assessment of the conidia yield in Neurospora crassa in response to treatment with different conidiation effectors. Depending on nitrogen source and intactness of nitrite reductase (NiR) and nitrate reductase (NR), light and dehydration affected the number of viable conidia produced by the ascomycete. In most variants of the nitrogen status, the combined action of light and dehydration synergistically increased the conidia yield. Conidiation in wild-type cells cultivated on the medium with NH4Cl as a sole nitrogen source did not respond to light, whereas illumination of the same culture grown on NH4NO3- or NaNO3-containing medium stimulated the process of spore formation. In response to light exposure, conidia formation occurred in the same way in the nit-2 (no NR and NiR) and nit-6 (no NiR) mutants cultivated in the presence of NH4Cl, but differed greatly when grown on the medium with NH4NO3. The results obtained indicate the possibility that NR and NiR participate in the photoconidiation regulation (wild-type strain on the medium with secondary nitrogen source); however, they cannot be necessary because light-dependent stimulation of spore formation was observed in nit-2 and nit-6 mutants.

A phylogenetic assessment and taxonomic revision of the thermotolerant hyphomycete genera Acrophialophora and Taifanglania.

We assessed the phylogenetic relationships of 19 isolates belonging to Acrophialophora and Taifanglania based on internal transcribed spacer (ITS), nuclear 18S subunit (nuc 18S) rDNA and ß-tubulin sequences. Phylogenetic data showed that Acrophialophora and Taifanglania comprise a monophyletic clade, but did not support the distinction of two genera. Being the older and more frequently used name, Acrophialophora is adopted as the generic name and Taifanglania is treated as a synonym. The generic concept of Acrophialophora is emended to include the morphological characters formerly used to distinguish Taifanglania. Three new thermotolerant species isolated from soil samples in China are described and illustrated, (i) A. ellipsoidea, with solitary phialides tapering into thin necks and long chains of ellipsoidal to fusiform conidia, (ii) A. angustiphialis with single phialides terminal or lateral on hyphae, and long chains of ellipsoidal or fusiform conidia and, (iii) A. acuticonidiata with single phialides and fusiform conidia with acute ends. Phylogenetic analyses show that A. acuticonidiata, A. angustiphialis and A. ellipsoidea are most closely related to A. curticatenata, A. hechuanensis and A. major, respectively. Growth tests showed that the three new species are thermotolerant, with optimal growth temperatures of 37-40 C, and maximum growth temperatures near 50 C. A key to the accepted species of Acrophialophora is provided.

Systematics of the Trichoderma harzianum species complex and the re-identification of commercial biocontrol strains.

Trichoderma harzianum is known as a cosmopolitan, ubiquitous species associated with a wide variety of substrates. It is possibly the most commonly used name in agricultural applications involving Trichoderma, including biological control of plant diseases. While various studies have suggested that T. harzianum is a species complex, only a few cryptic species are named. In the present study the taxonomy of the T. harzianum species complex is revised to include at least 14 species. Previously named species included in the complex are T. guizhouense, T. harzianum, and T. inhamatum. Two new combinations are proposed, T. lentiforme and T. lixii. Nine species are described as new, T. afarasin, T. afroharzianum, T. atrobrunneum, T. camerunense, T. endophyticum, T. neotropicale, T. pyramidale, T. rifaii and T. simmonsii. We isolated Trichoderma cultures from four commercial biocontrol products reported to contain T. harzianum. None of the biocontrol strains were identified as T. harzianum s. str. In addition, the widely applied culture 'T. harzianum T22' was determined to be T. afroharzianum. Some species in the T. harzianum complex appear to be exclusively endophytic, while others were only isolated from soil. Sexual states are rare. Descriptions and illustrations are provided. A secondary barcode, nuc translation elongation factor 1-α (TEF1) is needed to identify species in this complex.

The jack of all trades is master of none: a pathogen's ability to infect a greater number of host genotypes comes at a cost of delayed reproduction.

A trade-off between a pathogen's ability to infect many hosts and its reproductive capacity on each host genotype is predicted to limit the evolution of an expanded host range, yet few empirical results provide evidence for the magnitude of such trade-offs. Here, we test the hypothesis for a trade-off between the number of host genotypes that a fungal pathogen can infect (host genotype range) and its reproductive capacity on susceptible plant hosts. We used strains of the oat crown rust fungus that carried widely varying numbers of virulence (avr) alleles known to determine host genotype range. We quantified total spore production and the expression of four pathogen life-history stages: infection efficiency, time until reproduction, pustule size, and spore production per pustule. In support of the trade-off hypothesis, we found that virulence level, the number of avr alleles per pathogen strain, was correlated with significant delays in the onset of reproduction and with smaller pustule sizes. Modeling from our results, we conclude that trade-offs have the capacity to constrain the evolution of host genotype range in local populations. In contrast, long-term trends in virulence level suggest that the continued deployment of resistant host lines over wide regions of the United States has generated selection for increased host genotype range.

Air analysis in the assessment of fumonisin contamination risk in maize.

BACKGROUND: In maize-growing areas where fumonisin contamination is endemic, there is an urgent need for novel methods to assess the quality of grain lots before their delivery to common drying and storage collection centres. Aerobiological samples of fungal spores released during harvest were analysed to establish a relationship between fumonisin contamination and the abundance of pathogen propagules collected in the combine harvester using a cyclone and membrane filters. Filter-captured propagules were analysed by direct plating, immunoenzymatic assay of specific Fusarium extracellular polysaccharides and real time polymerase chain reaction of the extracted DNA using fum1, a gene involved in the biosynthesis of fumonisin, as a target. RESULTS: The results showed that time of harvest and environmental conditions strongly influenced the efficiency and performance of the collection system. The data obtained were informative in comparing individual samples collected under similar conditions. The immunoenzymatic assay provided the most reliable data, which improved the ability of a neural network to predict the fumonisin content of lots, when added to agronomic, environmental and phytosanitary data. CONCLUSION: This is the first attempt to evaluate the Fusarium propagules dispersed during harvesting as a predictive means to assess maize quality. A method based on cyclone/filter capture and immunological detection has been shown to be feasible and to have the potential for the development of a continuous monitoring system, but the prediction capabilities in the present implementation were limited.