Exploring structural and molecular diversity of Ericaceae hair root mycobionts: a comparison between Northern Bohemia and Argentine Patagonia.

Core Ericaceae produce delicate hair roots with inflated rhizodermal cells that host plethora of fungal symbionts. These poorly known mycobionts include various endophytes, parasites, saprobes, and the ericoid mycorrhizal (ErM) fungi (ErMF) that form the ErM symbiosis crucial for the fitness of their hosts. Using microscopy and high-throughput sequencing, we investigated their structural and molecular diversity in 14 different host × site combinations in Northern Bohemia (Central Europe) and Argentine Patagonia (South America). While we found typical ericoid mycorrhiza in all combinations, we did not detect ectomycorrhiza and arbuscular mycorrhiza. Superficial mantles of various thickness formed by non-clamped hyphae were observed in all combinations except Calluna vulgaris from N. Bohemia. Some samples contained frequent intercellular hyphae while others possessed previously unreported intracellular haustoria-like structures linked with intracellular hyphal coils. The 711 detected fungal OTU were dominated by Ascomycota (563) and Basidiomycota (119), followed by four other phyla. Ascomycetes comprised Helotiales (255), Pleosporales (53), Chaetothyriales (42), and other 19 orders, while basidiomycetes Sebacinales (42), Agaricales (28), Auriculariales (7), and other 14 orders. While many dominant OTU from both hemispheres lacked close relatives in reference databases, many were very similar to identical to unnamed sequences from around the world. On the other hand, several significant ericaceous mycobionts were absent in our dataset, incl. Cairneyella, Gamarada, Kurtia, Lachnum, and Leohumicola. Most of the detected OTU could not be reliably linked to a particular trophic mode, and only two could be reliably assigned to the archetypal ErMF Hyaloscypha hepaticicola. Probable ErMF comprised Hyaloscypha variabilis and Oidiodendron maius, both detected only in N. Bohemia. Possible ErMF comprised sebacinoid fungi and several unnamed members of Hyaloscypha s. str. While H. hepaticicola was dominant only in C. vulgaris, this model ErM host lacked O. maius and sebacinoid mycobionts. Hyaloscypha hepaticicola was absent in two and very rare in six combinations from Patagonia. Nine OTU represented dark septate endophytes from the Phialocephala fortinii s. lat.-Acephala applanata species complex, including the most abundant OTU (the only detected in all combinations). Statistical analyses revealed marked differences between N. Bohemia and Patagonia, but also within Patagonia, due to the unique community detected in a Valdivian temperate rainforest. Our results show that the ericaceous hair roots may host diverse mycobionts with mostly unknown functions and indicate that many novel ErMF lineages await discovery. Transhemispheric differences (thousands of km) in their communities may be evenly matched by local differences (scales of km, m, and less).

Evaluation and selection of culture media for the detection of auxin-like compounds and phosphate solubilization on soil yeasts.

The detection of characteristics associated with plant growth promotion has been studied frequently on bacteria and some of these detection methods were also used on yeasts. Sometimes, these methods, designed for prokaryotes, were used with no prior analysis regarding growth and production on eukaryotes. The aim of the present study was to assess and select the best suitable media for the detection of auxin-like compound production and inorganic-P solubilization on yeast strains. Already published media and new formulations, as well as yeasts with different genetic backgrounds were used for the comparison. Media were selected based on the adequate growth of yeast strains and reliable recognition of the studied features either by an easy detection of the metabolite (color or halo production) or simple medium preparation (low number of reagent and regular autoclaving). We propose here the use of the new DEV medium with glucose and with tryptophan to identify auxin-like compounds producers; and CPM medium (a variation from Custer's Chalk Medium) as base medium to identify yeasts capable of inorganic-P solubilization.

First record of Rhizoscyphus ericae in Southern Hemisphere's Ericaceae.

Ericoid mycorrhiza is arguably the least investigated mycorrhizal type, particularly when related to the number of potential hosts and the ecosystems they inhabit. Little is known about the global distribution of ericoid mycorrhizal (ErM) fungi, and this holds true even for the prominent ErM mycobiont Rhizoscyphus ericae. Earlier studies suggested R. ericae might be low in abundance or absent in the roots of Southern Hemisphere's Ericaceae, and our previous investigations in two Argentine Patagonian forests supported this view. Here, we revisited the formerly investigated area, albeit at a higher altitude, and screened fungi inhabiting hair roots of Gaultheria caespitosa and Gaultheria pumila at a treeless alpine site using the same methods as previously. We obtained 234 isolates, most of them belonging to Ascomycota. In contrast to previous findings, however, among 37 detected operational taxonomic units (OTUs), OTU 1 (=R. ericae s. str.) comprised the highest number of isolates (87, ∼37 %). Most of the OTUs and isolates belonged to the Helotiales, and 82.5 % of isolates belonged to OTUs shared between both Gaultheria species. At the alpine site, ericoid mycorrhizal fungi dominated, followed by dark septate endophytes and aquatic hyphomycetes probably acting as root endophytes. Our results suggest that the distribution of R. ericae is influenced, among others, by factors related to altitude such as soil type and presence/absence and type of the neighboring vegetation. Our study is the first report on R. ericae colonizing Ericaceae roots in the Southern Hemisphere and extends the known range of this prominent ErM species to NW Patagonia.

Detection of plant growth enhancing features in psychrotolerant yeasts from Patagonia (Argentina).

This study explores the biotechnological potential for plant production of twelve psychrotolerant yeasts strains from Northwest-Patagonia. These strains were isolated from different substrates associated with Nothofagus sp. in native forests and Vaccinium sp. in a commercial plantation. Yeasts characterization was performed using in vitro assays to evaluate the production of auxin-like compounds and siderophores, ability to solubilize inorganic phosphate and to reduce common plant pathogen growth. Strain YF8.3 identified as Aureobasidium pullullans was the main producer of auxin-like and siderophores compounds. Phosphate solubilization was a characteristic observed by strains L8.12 and CRUB1775 identified as Holtermaniella takashimae and Candida maritima, respectively. Different yeast strains were able to inhibit the growth of Verticillium dahliae PPRI5569 and Pythium aphanidermatum PPRI 9009, but they all failed to inhibit the growth of Fusarium oxysporum PPRI5457. The present study, suggests that yeasts present in different environments in Northwestern-Patagonian have physiological in vitro features which may influence plant growth. These results are promising for the developing of biological products based on Patagonian yeasts for plant production in cold-temperate regions.

Is the prominent ericoid mycorrhizal fungus Rhizoscyphus ericae absent in the Southern Hemisphere's Ericaceae? A case study on the diversity of root mycobionts in Gaultheria spp. from northwest Patagonia, Argentina.

Ericaceae diversity hotspots are in the mountains of the Neotropics and Papua New Guinea, South Africa's fynbos and Southeast Asia but majority of references to their root mycobionts come from the Northern Hemisphere. Here, typical cultivable ericoid mycorrhizal (ErM) fungi comprise Rhizoscyphus ericae, Meliniomyces variabilis, and Oidiodendron maius. It is however unclear whether this is true also for the Southern Hemisphere. Our study focused on cultivable mycobionts from hair roots of Gaultheria mucronata and Gaultheria poeppigii (Ericaceae) from two natural forests in NW Patagonia, Argentina, differing in mycorrhizal preferences of their tree dominants. We detected 62 well-defined OTUs mostly belonging to Helotiales and Hypocreales; the most frequent were Phialocephala fortinii s. l., Pochonia suchlasporia, and Ilyonectria radicicola. Only one out of 257 isolates showed ITS nrDNA similarity to members of the R. ericae aggregate (REA) but was not conspecific with R. ericae, and only five isolates were conspecific with O. maius. Microscopic observations showed that the screened roots were frequently colonized in a manner differing from the pattern typically produced by R. ericae and O. maius. A re-synthesis experiment with selected isolates showed that only O. maius formed colonization resembling ericoid mycorrhiza. Amplification of root fungal DNA with REA-specific and Sebacinaceae-specific primers showed that REA mycobionts were present in some of the screened samples while Sebacinaceae were present in all samples. These results suggest that Gaultheria spp. from NW Patagonia form ericoid mycorrhizae predominantly with the difficult-to-cultivate Sebacinaceae while the incidence of REA is relatively low and may be masked by other most likely non-mycorrhizal cultivable mycobionts.

Ecology of cultivable yeasts in pristine forests in northern Patagonia (Argentina) influenced by different environmental factors.

Environmental factors influencing the occurrence and community structure of soil yeasts in forests are not well studied. There are few studies dedicated to Southern Hemisphere soil yeasts populations and even fewer focused on temperate forests influenced by volcanic activity. The present work aimed to study the ecology of soil yeast communities from pristine forests influenced by different environmental factors (precipitation, physicochemical properties of soil, tree species, soil region, and season). The survey was performed in 4 northern Patagonian forests: 2 dominated by Nothofagus pumilio and 2 by Nothofagus antarctica. Yeast communities were described with ecological indices and species accumulation curves, and their association with environmental characteristics was assessed using multivariate analysis. Each forest site showed a particular arrangement of species as a result of environmental characteristics, such as dominant plant species, nutrient availability, and climatic characteristics. Cryptococcus podzolicus was most frequently isolated in nutrient-rich soils, Trichosporon porosum dominated cold mountain forests with low nutrient and water availability in soil, and capsulated yeasts such as Cryptococcus phenolicus dominated forest sites with low precipitation. The present work suggests that environmental factors affecting yeast communities may not be the current soil characteristics but the result of complex interactions of factors including natural disturbances like volcanic activity.

Ectomycorrhizas naturally established in Nothofagus nervosa seedlings under different cultivation practices in a forest nursery.

Mycorrhizas are mutualistic associations between soil fungi and plant roots which usually improve water and nutrient uptake, influencing plant fitness. Nothofagus nervosa (Raulí) is an ecologically and economically important species of South American temperate forests. Since this native tree species yields valuable timber, it was overexploited and its natural distribution area was critically reduced, so it is currently included in domestication and conservation programs. Among the factors that should be considered in these programs are the ectomycorrhizas (EcM), which would be important for the successful establishment and survival of outplanted seedlings. The aim of this work was to analyze the abundance and diversity of EcM in N. nervosa nursery-cultivated seedlings assessed by morphotyping, fungal isolation, and DNA sequencing. Arbuscular mycorrhiza (AM) occurrence was also studied. A 2-year trial was conducted following the cultivation conditions used for domestication programs. Seedlings were cultivated under two different cultivation practices (greenhouse and nursery soil) without artificial inoculation of mycorrhizal fungi. Seedlings' roots were examined at different times. It was observed that they developed EcM between 6 and 12 months after germination and AMs were not detected in any plant. The most abundant ectomycorrhizal fungi present in seedlings' roots were Tomentella ellisii (Basidiomycota) and an unidentified fungus named Ascomicetous EcM sp. 1. Abundance and diversity of EcM varied between the two cultivation techniques analyzed in this study, since seedlings that continued growing in the greenhouse had higher colonization values, but those transplanted to the nursery soil were colonized by a higher diversity of fungal taxa.

Occurrence of arbuscular mycorrhizas and dark septate endophytes in pteridophytes from a Patagonian rainforest, Argentina.

Arbuscular mycorrhizas (AM) are one of the most widespread types of symbiotic associations. Pteridophytes occupy an important position in the evolution of vascular plants. However, their mycorrhizal state remains poorly understood. The aim of this work was to describe the general mycorrhizal status and the occurrence of dark septate endophytes (DSE) in the pteridophytic flora of a Valdivian temperate forest in Patagonia, Argentina. First, the roots of nine terrestrial species representing six families were examined, and this information was then compared with other surveys concerning the occurrence of AM in other pteridophytic species within the same Valdivian temperate forest. AM were recorded in 98.6% of the samples analyzed in this work and all of them corresponded to the Paris-type morphology. DSEs were also present within the roots of all terrestrial species. A comparison to published results in other ferns and lycophytes that have been studied in this Valdivian temperate forest (161 sporophytes, 21 species and 10 families) was made. Clear differences in colonization patterns between eusporangiate/leptosporangiate and epiphytic/terrestrial species became evident and are discussed.

Differential hypogeous sporocarp production from Nothofagus dombeyi and N. pumilio forests in southern Argentina.

Mycorrhizal fungi that form hypogeous sporocarps are an important component of the temperate forest soil community. In many regions, such as the Nothofagus forest in the Patagonian Andes, this group of fungi has been poorly studied. Here we examined the spring and autumn community composition of "sequestrate fungi", based on sporocarp production in pure forests of Nothofagus dombeyi (evergreen) and N. pumilio (deciduous). We investigated the possible relationships between these communities and environmental factors over 2 y. The rarefaction curves and the minimal richness estimates converged at nearly the same level for each forest type, and the asymptotes suggested that the sampling effort was sufficient to capture most of the hypogeous sporocarp richness in these forest stands. In total 27 species were recovered. Basidiomycota, Ascomycota and Glomeromycota respectively accounted for nine, two and one genera. Species richness of hypogeous sporocarps varied in relation to forest type but not to season (fall and spring), whereas sporocarp biomass varied according to an interaction between season and forest type. Species richness and sporocarp biomass were positively correlated with rainfall and negatively correlated with altitude. In addition sporocarp species richness was positively related to number of trees per transect. We found that two different forest stands, each dominated by different species of Nothofagus, exhibited different hypogeous sporocarp communities.

Lindnera rhizosphaerae sp. nov., a yeast species isolated from rhizospheric soil.

Two strains of a novel yeast species were isolated from ectomycorrhizal Nothofagus pumilio rhizospheric soil in a native forest of Patagonia, Argentina. Analysis of the D1/D2 large-subunit rRNA gene sequences indicated that the novel species belongs to the recently described genus Lindnera. The name Lindnera rhizosphaerae sp. nov. is proposed to accommodate these isolates, and the type strain is CRUB 1796(T) ( = CBS 11400(T)  = JCM 16499(T)).

Yeast and yeast-like fungi associated with dry indehiscent fruits of Nothofagus nervosa in Patagonia, Argentina.

Nothofagus nervosa (Raulí) is a native tree species that yields valuable timber. It was overexploited in the past and is currently included in domestication and conservation programs. Several research programs have focused on the characterization of epiphytic microorganisms because it has been demonstrated that they can affect plant-pathogen interactions and/or promote plant growth. Although the microbial ecology of leaves has been well studied, less is known about microorganisms occurring on seeds and noncommercial fruits. In this work, we analyzed the yeast and yeast-like fungi present on N. nervosa fruits destined for the propagation of this species, as well as the effects of fruit preservation and seed dormancy-breaking processes on fungal diversity. Morphological and molecular methods were used, and differences between fungal communities were analyzed using a similarity index. A total of 171 isolates corresponding to 17 species were recovered, most of which belong to the phylum Ascomycota. The majority of the species develop mycelia, produce pigments and mycosporines, and these adaptation strategies are discussed. It was observed that the preservation process considerably reduced yeast and yeast-like fungal diversity. This is the first study concerning microbial communities associated with this ecologically and economically important species, and the information presented is relevant to domestication programs.

Yeast communities associated with the bulk-soil, rhizosphere and ectomycorrhizosphere of a Nothofagus pumilio forest in northwestern Patagonia, Argentina.

Soil microorganisms play an important role in soil quality and they interact closely with vegetation. Little is known about yeast diversity and function in forest soil ecosystems and their interactions with other biotic soil components, particularly in the mycorrhizosphere. We studied the diversity of yeasts inhabiting the bulk-soil, rhizosphere and ectomycorrhizosphere of a Nothofagus pumilio forest in Nahuel Huapi National Park (Bariloche, Argentina). Ectomycorrhizal infection was observed in all N. pumilio trees studied. A total of 126 yeast isolates were obtained, including 18 known and three possibly new species. Basidiomycetous yeasts were predominant in all soil fractions, and the most frequently isolated species was Cryptococcus podzolicus. Diversity indices and multivariate analyses were used to study and compare yeast communities in the bulk-soil, rhizosphere and ectomycorrhizosphere. Yeasts able to ferment glucose were found associated with the rhizosphere. Many of the recovered yeast species were associated with lignocelluloses compound degradation, which suggest that yeast plays an important role as a decomposer in these forest soils. Each soil fraction has a distinct yeast assemblage related to their physiologic capacities and soil nutrient availability.

Characterization of a novel South American population of the astaxanthin producing yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

A novel population of the biotechnologically important yeast Xanthophyllomyces dendrorhous, the sexual stage of Phaffia rhodozyma, has been recently isolated for the first time in the southern Hemisphere (Patagonia, Argentina). The aim of the present work was to phenotypically and genotypically characterize two representative strains of this new population, and assess such strains as a potential biotechnological source of astaxanthin, fatty acids and extracellular enzymes. Minor variations were found in physiological tests. PCR fingerprinting studies (MSP-PCR) showed the main differences between X. dendrorhous Patagonian and Type strains. Patagonian strains accumulated a xanthophyll-like pigment, which was identified as astaxanthin. These strains showed low fatty acids content (mainly polyunsaturated fatty acids) and, of a total of six extracellular enzymes tested, only produced amylase. Genetic differences between Patagonian and collection X. dendrorhous strains could be explained by geographic isolation and habitat specificity.

Infectivity of soilborne Frankia and mycorrhizae in Discaria trinervis along a vegetation gradient in Patagonian soil.

The infective capacities of the nitrogen fixing Actinomycete Frankia and arbuscular mycorrhizal fungi from soils near watercourses, along a vegetation gradient, were studied using plant bioassays. Frankia and arbuscular mycorrhizas capable of infecting Discaria trinervis were found at seventeen sites sampled. More specific enumeration of the infective capacities of both microorganisms in relation to environmental factors was performed in seven representative soils of the analysed vegetation zones (rainforest, xeric forest and steppe) using the most probable number method. The highest nodulation capacities ranged from 340 infective units g(-1 )soil, in a steppe marsh devoid of actinorhizas, to 61 in a coastal actinorhizal scrub (in xeric forest). The highest number of infective mycorrhizal units--also found in marsh--was 145. In general, rainforest soils had the lowest values for both microorganisms. Infective units of Frankia and arbuscular mycorrhizal fungi in soil were positively correlated (r = 0.89, P < 0.05). Both soilborne symbionts showed the highest infective capacity in semi-arid conditions nearby watercourse and at the valley bottom location. Tripartite symbiosis was effective in plants inoculated with steppe and xeric forest soils and plants inoculated with Frankia BCU110501 and Glomus mosseae. Interaction between both symbionts and influence of environmental conditions, in general, would contribute to define comparable trends of their infective capacities.

Estudio de confiabilidad de dos técnicas para el inmunodiagnóstico de la enfermedad de Chagas / Confiability study of 2 techniques for inmunodiagnosis of Chagas' disease

Ante la dificultad de aislar el parásito en la infección chagásica crónica, la detección de anticuerpos circulantes es, con frecuencia, el único parámetro que permite realizar el diagnóstico y encuestas seroepidemiológicas. Por ello y por la creciente preocupación acerca de la validez de los resultados, se realizó este trabajo para evaluar la confiabilidad de dos técnicas serológicas muy utilizadas, Aglutinación Directa y Hemaglutinación Indirecta. Se emplearon parámetros de control de calidad. De los resultados obtenidos se concluye que ambas técnicas poseen elevados índices de Sensibilidad, Especificidad y Precisión; y que los valores predictivos (positivos y negativos) y Eficiencia aumentan cuando se realizan ambas técnicas en conjunto

Estudio de confiabilidad de dos técnicas para el inmunodiagnóstico de la enfermedad de Chagas / Confiability study of 2 techniques for inmunodiagnosis of Chagas disease

Ante la dificultad de aislar el parásito en la infección chagásica crónica, la detección de anticuerpos circulantes es, con frecuencia, el único parámetro que permite realizar el diagnóstico y encuestas seroepidemiológicas. Por ello y por la creciente preocupación acerca de la validez de los resultados, se realizó este trabajo para evaluar la confiabilidad de dos técnicas serológicas muy utilizadas, Aglutinación Directa y Hemaglutinación Indirecta. Se emplearon parámetros de control de calidad. De los resultados obtenidos se concluye que ambas técnicas poseen elevados índices de Sensibilidad, Especificidad y Precisión; y que los valores predictivos (positivos y negativos) y Eficiencia aumentan cuando se realizan ambas técnicas en conjunto (AU)