Contribution of Arbuscular Mycorrhizal and Endophytic Fungi to Drought Tolerance in Araucaria araucana Seedlings.

In its natural distribution, Araucaria araucana is a plant species usually exposed to extreme environmental constraints such as wind, volcanism, fires, and low rainfall. This plant is subjected to long periods of drought, accentuated by the current climate emergency, causing plant death, especially in its early growth stages. Understanding the benefits that both arbuscular mycorrhizal fungi (AMF) and endophytic fungi (EF) could provide plants under different water regimes would generate inputs to address the above-mentioned issues. Here, the effect of AMF and EF inoculation (individually and combined) on the morphophysiological variables of A. araucana seedlings subjected to different water regimes was evaluated. Both the AMF and EF inocula were obtained from A. araucana roots growing in natural conditions. The inoculated seedlings were kept for 5 months under standard greenhouse conditions and subsequently subjected to three different irrigation levels for 2 months: 100, 75, and 25% of field capacity (FC). Morphophysiological variables were evaluated over time. Applying AMF and EF + AMF yielded a noticeable survival rate in the most extreme drought conditions (25% FC). Moreover, both the AMF and the EF + AMF treatments promoted an increase in height growth between 6.1 and 16.1%, in the production of aerial biomass between 54.3 and 62.6%, and in root biomass between 42.5 and 65.4%. These treatments also kept the maximum quantum efficiency of PSII (Fv/Fm 0.71 for AMF and 0.64 for EF + AMF) stable, as well as high foliar water content (>60%) and stable CO2 assimilation under drought stress. In addition, the EF + AMF treatment at 25% FC increased the total chlorophyll content. In conclusion, using indigenous strains of AMF, alone or in combination with EF, is a beneficial strategy to produce A. araucana seedlings with an enhanced ability to tolerate prolonged drought periods, which could be of great relevance for the survival of these native species under the current climate change.

Molecular and cultural characterization of Morchella spp. from disturbed environments of central-southern Chile.

In Northwestern Patagonia (Chile), three species of Morchella from undisturbed environments have been identified to date: Morchella tridentina, Morchella andinensis and Morchella aysenina, all belonging to the Elata clade and associated mainly with Nothofagus forests. In this study, the search for Morchella specimens was extended to disturbed environments in Central-Southern Chile, to further explore Morchella species diversity in the country, which is still very limited. The Morchella specimens were identified through multilocus sequences analysis, and the mycelial cultures were characterized, establishing comparisons with specimens from undisturbed environments. To the best of our knowledge, these results reveal for the first time in Chile the presence of the species Morchella eximia and Morchella importuna, and in the case of the last one also the first record in South America. These species were found associated almost exclusively with harvested or burned coniferous plantations. The in vitro mycelial characterization revealed certain inter- and intra-specific patterns of the morphology, such as pigmentation, mycelium type, and development and formation of sclerotia, which varied according to growth media and incubation temperature. The growth rates (mm/day) and mycelial biomass (mg) were significantly influenced by the temperature (p < 0.05), with maximum rates (>10 mm/day) and biomass (approx. 20 mg) between 20 and 24 °C, while a significant growth reduction (70-90%) was observed at 28 °C, mainly in the species from undisturbed environments. Potato-dextrose (PDA) medium stimulated the greatest mycelial density and sclerotia formation in most of the isolates, mainly in M. eximia (UDEC-LAF 236 isolate) which recorded the best mycelial growth performance. Among isolates, UDEC-LAF 236 also showed the best performance in sclerotia production (>350 sclerotia/dish) in 10 days of growth. This study contributes to the knowledge of the diversity of Morchella species in Chile by broadening the species range to those from disturbed environments. It also provides molecular and morphological characterization of the in vitro cultures of different Morchella species. The report on M. eximia and M. importuna, species known as cultivable, adapted to local climatic and edaphic conditions could represent the first step to developing artificial Morchella cultivation methods in Chile.

Genetic Study of SARS-CoV-2 Non Structural Protein 12 in COVID-19 Patients Non Responders to Remdesivir.

Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (n = 36/111, 32.4%) or prolonged (5 to 20 days) (n = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the nsp12 gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the de novo E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. IMPORTANCE This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the nsp12 pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.

Chemical characterization and microencapsulation of extracellular fungal pigments.

In this work, extracellular colored metabolites obtained from the filamentous fungi Talaromyces australis and Penicillium murcianum, isolated in the Andean-Patagonian native forests of Chile, were studied as prospect compounds to increase the sustainability of cosmetic products. The chemical and antioxidant properties of these natural pigments were characterized and strategies for their microencapsulation were also studied. UHPLC/MS-MS analyses indicated that the predominant metabolites detected in the cultures of P. murcianum were monascin (m/z = 411.15) and monashexenone (m/z = 319.10), while athrorosin H (m/z = 458.20) and damnacanthal (m/z = 281.05) were detected in cultures of T. australis. ORAC tests revealed that P. murcianum's metabolites had the greatest antioxidant properties with values higher than 2000 µmol of trolox equivalents/g. The fungal metabolites were successfully microencapsulated by ionic gelation into structures made of 1.3% sodium alginate, 0.2% chitosan, and 0.07% hyaluronic acid. The microencapsulation process generated structures of 543.57 ± 0.13 µm of mean diameter (d50) with an efficiency of 30% for P. murcianum, and 329.59 ± 0.15 µm of mean diameter (d50) and 40% efficiency, for T. australis. The chemical and biological characterization show the biotechnological potential of these fungal species to obtain pigments with antioxidant activity that could be useful in the cosmetic industry. The encapsulation process enables the production of easy-to-handle dry powder from the fungal metabolites, which could be potentially marketed as a functional cosmetic ingredient. KEY POINTS: • The predominant fungal pigments were of azaphilone and anthraquinoid classes. • The fungal pigments showed high antioxidant activity by ORAC assay. • Fungal pigment microcapsules obtained by ionic gelation were characterized.

Alkalization of Kraft Pulps from Pine and Eucalyptus and Its Effect on Enzymatic Saccharification and Viscosity Control of Cellulose.

Bleached kraft pulps from eucalyptus and pine were subjected to cold caustic extraction (CCE) with NaOH (5, 10, 17.5, and 35%) for hemicelluloses removal and to increase cellulose accessibility. The effect of these changes was evaluated in enzymatic saccharification with the multicomponent Cellic CTec3 enzyme cocktail, and in viscosity reduction of pulps with the monocomponent Trichoderma reesei endoglucanase (EG). After CCE with 10% NaOH (CCE10) and 17.5% NaOH (CCE17.5), hemicellulose content lower than 1% was achieved in eucalyptus and pine pulps, respectively. At these concentrations, cellulose I started to be converted into cellulose II. NaOH concentrations higher than 17.5% decreased the intrinsic viscosity (from 730 to 420 mL/g in eucalyptus and from 510 to 410 mL/g in pine). Cellulose crystallinity was reduced from 60% to 44% in eucalyptus and from 71% to 44% in pine, as the NaOH concentration increased. Enzymatic multicomponent saccharification showed higher glucose yields in all CCE-treated eucalyptus samples (up to 93%) while only CCE17.5 and CCE35 pine pulps achieved 90% after 40 h of incubation. Untreated bleached pulps of both species presented saccharification yields lower than 70%. When monocomponent EG was used to treat the same pulps, depending on enzyme charge and incubation time, a wide range of intrinsic viscosity reduction was obtained (up to 74%). Results showed that eucalyptus pulps are more accessible and easier to hydrolyze by enzymes than pine pulps and that the conversion of cellulose I to cellulose II hydrate only has the effect of increasing saccharification of CCE pine samples. Viscosity reduction of CCE pulps and EG treated pulps were obtained in a wide range indicating that pulps presented characteristics suitable for cellulose derivatives production.

Talaromyces australis and Penicillium murcianum pigment production in optimized liquid cultures and evaluation of their cytotoxicity in textile applications.

In this work Talaromyces australis and Penicillium murcianum pigment production in liquid cultures and the cytotoxic effect of such pigments on skin model cells were studied. Response surface methodology (RSM) was used to optimize culture conditions aiming to increase pigment production in malt extract and peptone-glucose-yeast extract medium. Cytotoxicity of fungal pigments and also from lixiviates of wool fabrics dyed with T. australis and P. murcianum pigment was evaluated on mammalian cell lines HEK293 and NIH/3T3. Results showed that variations on initial pH, NaCl and peptone, resulted in increments up to 188.2% for red pigment of T. australis and 107.4% for yellow pigment of P. murcianum, regarding non-optimized conditions. Tested fungi also showed great differences in culture conditions for the maximum pigment production, with P. murcianum requiring an alkaline medium (initial pH 9) supplemented with NaCl and T. australis an acidic medium (initial pH 5) without addition of salt. The cytotoxicity assays provided evidences on the safe nature of these natural pigments when used for textile applications. The cytotoxicity assay showed that the threshold of toxicity, given by the lowest IC50 value (0.21 g L-1) was more than double of the concentration of pigment required to dye the wool samples. In addition, cytotoxicity of lixiviates depicted no toxic effect over tested cells.

Redox-dependent and redox-independent functions of Caenorhabditis elegans thioredoxin 1.

Thioredoxins (TRX) are traditionally considered as enzymes catalyzing redox reactions. However, redox-independent functions of thioredoxins have been described in different organisms, although the underlying molecular mechanisms are yet unknown. We report here the characterization of the first generated endogenous redox-inactive thioredoxin in an animal model, the TRX-1 in the nematode Caenorhabditis elegans. We find that TRX-1 dually regulates the formation of an endurance larval stage (dauer) by interacting with the insulin pathway in a redox-independent manner and the cGMP pathway in a redox-dependent manner. Moreover, the requirement of TRX-1 for the extended longevity of worms with compromised insulin signalling or under calorie restriction relies on TRX-1 redox activity. In contrast, the nuclear translocation of the SKN-1 transcription factor and increased LIPS-6 protein levels in the intestine upon trx-1 deficiency are strictly redox-independent. Finally, we identify a novel function of C. elegans TRX-1 in male food-leaving behaviour that is redox-dependent. Taken together, our results position C. elegans as an ideal model to gain mechanistic insight into the redox-independent functions of metazoan thioredoxins, overcoming the limitations imposed by the embryonic lethal phenotypes of thioredoxin mutants in higher organisms.

Cardiac, mandibular and thymic phenotypical association indicates that cranial neural crest underlies bicuspid aortic valve formation in hamsters.

Bicuspid aortic valve (BAV) is the most prevalent human congenital cardiac malformation. It may appear isolated, associated with other cardiovascular malformations, or forming part of syndromes. Cranial neural crest (NC) defects are supposed to be the cause of the spectrum of disorders associated with syndromic BAV. Experimental studies with an inbred hamster model of isolated BAV showed that alterations in the migration or differentiation of the cardiac NC cells in the embryonic cardiac outflow tract are most probably responsible for the development of this congenital valvular defect. We hypothesize that isolated BAV is not the result of local, but of early alterations in the behavior of the NC cells, thus also affecting other cranial NC-derived structures. Therefore, we tested whether morphological variation of the aortic valve is linked to phenotypic variation of the mandible and the thymus in the hamster model of isolated BAV, compared to a control strain. Our results show significant differences in the size and shape of the mandible as well as in the cellular composition of the thymus between the two strains, and in mandible shape regarding the morphology of the aortic valve. Given that both the mandible and the thymus are cranial NC derivatives, and that the cardiac NC belongs to the cephalic domain, we propose that the causal defect leading to isolated BAV during embryonic development is not restricted to local alterations of the cardiac NC cells in the cardiac outflow tract, but it is of pleiotropic or polytopic nature. Our results suggest that isolated BAV may be the forme fruste of a polytopic syndrome involving the cranial NC in the hamster model and in a proportion of affected patients.

Using common mycorrhizal networks for controlled inoculation of Quercus spp. with Tuber melanosporum: the nurse plant method.

The high cost and restricted availability of black truffle spore inoculum for controlled mycorrhiza formation of host trees produced for truffle orchards worldwide encourage the search for more efficient and sustainable inoculation methods that can be applied globally. In this study, we evaluated the potential of the nurse plant method for the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum by mycorrhizal networks in pot cultures. Pine bark compost, adjusted to pH 7.8 by liming, was used as substrate for all assays. Initially, Q. robur seedlings were inoculated with truffle spores and cultured for 12 months. After this period, the plants presenting 74 % mycorrhizal fine roots were transferred to larger containers. Nurse plants were used for two treatments of two different nursling species: five sterilized acorns or five 45-day-old, axenically grown Q. robur or Q. cerris seedlings, planted in containers around the nurse plant. After 6 months, colonized nursling plant root tips showed that mycorrhiza formation by T. melanosporum was higher than 45 % in the seedlings tested, with the most successful nursling combination being Q. cerris seedlings, reaching 81 % colonization. Bulk identification of T. melanosporum mycorrhizae was based on morphological and anatomical features and confirmed by sequencing of the internal transcribed spacer region of the ribosomal DNA of selected root tips. Our results show that the nurse plant method yields attractive rates of mycorrhiza formation by the Périgord black truffle and suggest that establishing and maintaining common mycorrhizal networks in pot cultures enables sustained use of the initial spore inoculum.

Tolerance to wood preservatives by copper-tolerant wood-rot fungi native to south-central Chile.

Understanding the effect of heavy metals and wood preservatives on the growth of wood-rot fungi native to a certain region may improve reliability in determining the effectiveness of antifungal products, particularly when dealing with new formulations. In this investigation, strains of copper-tolerant wood-rot fungi native to south-central Chile were evaluated against two preservatives: commercial chromated copper arsenate type C (CCA-C) and a new formulation with boron and silicon (BS). Thirteen native strains, mainly white-rot fungi, were selected for their high growth rates in solid medium containing 3 mM of copper. A short-term test was then carried out, consisting of adding cellulose disks impregnated with different concentrations of preservatives to solid culture media inoculated with selected copper tolerant strains. There was a great variability in interspecific and intraspecific responses to the presence of copper and preservatives in culture media. Among the native and commercial strains evaluated, the white-rot fungi Trametes versicolor 38 and mainly Ganoderma australe 100 were notable for their tolerance to all the CCA-C and BS concentrations. The brown-rot fungus Wolfiporia cocos, used as reference strain, showed a high tolerance to CCA-C, but not to BS preservative. T. versicolor 38 and G. australe 100 were selected for subsequent studies on preserved wood degradation.