Natalia V. Fernández

How to know the fungi: combining field inventories and DNA‐barcoding to document fungal diversity

The fungi kingdom is among the most diverse eukaryotic lineages on Earth with estimates of several million extant species (O’Brien et al., 2005; Blackwell, 2011; Taylor et al., 2014). Fungi play critical roles in carbon andnutrient cycling of terrestrial and aquatic ecosystems, and they are important pathogens and mutualists (Read & Perez-Moreno, 2003; Taylor et al., 2012; Grossart et al., 2016). More than 80% of plant species form symbioses with fungi and these symbioses have been crucial to the colonization of terrestrial ecosystems (Field et al., 2015a; Selosse et al., 2015). Despite their impacts on primary ecosystem functions, assessments of fungal biodiversity estimate that only c. 10% of fungal species have been described (Bass & Richards, 2011; Hibbett et al., 2011). Traditionally, specimen-based taxonomic studies have been the only way to discover new species. Because most fungi have microscopic life-stages and convergent morphological features (Rivas-Plata & Lumbsch, 2011; Wynns, 2015), many fungal groups remain severely undersampled. DNA-barcoding and highthroughput sequencing methods have provided a new framework for studying fungal biodiversity (Fierer et al., 2012; Schoch et al., 2012; Myrold et al., 2014), and diversity estimates based on environmental sequences have increased exponentially. Although these ‘sequence-based classification and identification’ methods are a powerful means to rapidly detect hidden diversity, careful interpretation of these data is needed to make accurate inferences (K~oljalg et al., 2013; Lindahl et al., 2013; Nguyen et al., 2015; Hibbett et al., 2016). In particular, many environmental sequences cannot be associated with a known fungal species or lineage. This remains a major challenge to decipher fungal community composition and understand ecological roles of fungi in leaf litter, soil, or inside plants (Yahr et al., 2016). In some cases, these fungi are truly undescribed and their ecological roles are unknown but in other cases they represent described taxa for which no sequence is available (Nagy et al., 2011; Nilsson et al., 2016). DNA barcoding of herbarium specimens and culture collections is extremely valuable to link unidentified sequences to known taxa (e.g. Brock et al., 2009; Nagy et al., 2011; Osmundson et al., 2013; Garnica et al., 2016).DNA sequences have been generated from fungal type specimens > 200 years old (Larsson & Jacobsson, 2004), but in many cases obtaining sequences from historical material is challenging (Dentinger et al., 2010). Today’s threats to biodiversity from habitat loss and climate change are occurring at an unprecedented scale, and it is possible that many species may become extinct before they have been discovered (Costello et al., 2013; Monastersky, 2014). In the need to describe and protect as many species as possible we addressed the following questions: what are the best methods to rapidly document fungal biodiversity? Are traditional, specimen-based approaches still useful?

Mycorrhizal Status of Obligate and Facultative Epiphytic Ferns in a Valdivian Temperate Forest of Patagonia, Argentina

Abstract Arbuscular mycorrhizae (AM) influence the growth, morphology and fitness of the plant species they colonize. Despite the abundance and importance of epiphytes in forest dynamics, little is known about AM in these plants. Abundant epiphytes are present in the Valdivian temperate forests of South America, where ferns are one of the most important components of the epiphytic vascular flora. The aim of this work was to analyze the occurrence of AM in sporophytes of obligate and facultative epiphytic fern species in a Valdivian temperate forest of Patagonia, Argentina. We examined the roots of 10 epiphytic fern species. Roots of 66 sporophytes were collected from tree trunks and branches between 0.5 and 2 m height above the ground and were stained by using a modified Phillips and Hayman (1970) method. Of the 10 species examined in the present study two were found to be obligate epiphytes, six were facultative epiphytes and two could not be classified because few individuals were found. Most of them lacked AM structures, except for seven Hymenophyllum sporophytes which were rooted in soil and possessed scarce knobby hyphae and structures similar to coils and vesicles. Abundant dark septate fungi were observed within the cortex of all samples. The absence of AM in epiphytes may be explained by different ecological or taxonomic reasons, but in this study we suggest that root morphology may be an important factor influencing mycorrhizal behavior in ferns.

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.

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.

AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system

Angiotensin II (AngII), the main effector peptide of the renin‐angiotensin system (RAS), participates in multiple biological processes, including cell growth, apoptosis, and tissue remodeling. Since AngII activates, in different cell types, signal transducing pathways that are critical for mammary gland postlactational regression, we investigated the role of the RAS during this process. We found that exogenous administration of AngII in mammary glands of lactating Balb/c mice induced epithelium apoptosis [2.9±0.5% (control) vs. 9.6±1.1% (AngII); P < 0.001] and activation of the proapoptotic factor STAT3, an effect inhibited by irbesartan, an AT1 receptor blocker. Subsequently, we studied the expression kinetics of RAS components during involution. We found that angiotensin‐converting enzyme (ACE) mRNA expression peaked 6 h after weaning (5.7‐fold; P<0.01), while induction of angiotensinogen and AT1 and AT2 receptors expression was detected 96 h after weaning (6.2‐, 10‐, and 6.2‐fold increase, respectively; P<0.01). To assess the role of endogenously generated AngII, mice were treated with losartan, an AT1 receptor blocker, during mammary involution. Mammary glands from losartan‐treated mice showed activation of the survival factors AKT and BCL‐XL, significantly lower LIF and TNF‐α mRNA expression (P<0.05), reduced apoptosis [12.1±2.1% (control) vs. 4.8±0.7% (losartan); P<0.001] and shedding of epithelial cells, inhibition of MMP‐9 activity in a dose‐dependent manner (80%; P<0.05; with losartan IC50 value of 6.9 mg/kg/d] and lower collagen deposition and adipocyte invasion causing a delayed involution compared to vehicle‐treated mice. Furthermore, mammary glands of forced weaned AT1A‐ and/or AT1B‐deficient mice exhibited retarded apoptosis of epithelial cells [6.3±0.95% (WT) vs. 3.3±0.56% (AT1A/AT1B DKO); P<0.05] with remarkable delayed postlactational regression compared to wild‐type animals. Taken together, these results strongly suggest that AngII, via the AT1 receptor, plays a major role in mouse mammary gland involution identifying a novel role for the RAS.—Nahmod, K. A., Walther, T., Cambados, N., Fernandez, N., Meiss, R., Tappenbeck, N., Wang, Y., Raffo, D., Simian, M., Schwiebs, A., Pozner, R. G., Fuxman Bass, J. I., Pozzi, A. G., Geffner, J. R., Kordon, E. C., Schere‐Levy, C. AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system. FASEB J. 26, 1982‐1994 (2012). www.fasebj.org