MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation.

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

Differences in aluminum tolerance and immobilization between two indigenous ectomycorrhizal fungi Lactarius deliciosus and Pisolithus tinctorius from Southwest China's forest stands.

Aluminum (Al) toxicity severely decreases plant growth and productivity in acidic soil globally. Ectomycorrhizal (ECM) fungi can promote host plant's Al-tolerance by acting as a physical barrier or bio-filter. However, little information is available on the role of ECM fungus on Al immobilization with respect to Al-tolerance. This present study aimed to screen a promising indigenous ECM fungus with high Al-tolerance and to understand its role in Al immobilization related to Al-tolerance. Two ECM fungal strains (Lactarius deliciosus 2 and Pisolithus tinctorius 715) isolated from forest stands in Southwest China were cultured in vitro with 0.0, 1.0 or 2.0 mM Al addition for 21 days to compare their Al accumulation and Al-tolerance. Meanwhile, fungal mycelia were incubated in 0.037 mM Al3+ solutions, and then Al3+ concentrations in the solution were determined at time 2, 5, 10, 20, 40, 60, 120, 180, and 240 min, and the Al3+ immobilization characteristics were evaluated using the pseudo-first order, pseudo-second order and intraparticle diffusion models. Results showed that 1.0 or 2.0 mM Al3+ addition significantly increased fungal biomass production by 23% or 41% in L. deliciosus 2, not in P. tinctorius 715. Fungal Al3+ concentrations in L. deliciosus 2 and P. tinctorius 715 were significantly increased by 293% and 103% under 2.0 mM than under 1.0 mM Al3+ addition. The pH values in the culture solution were significantly decreased by 0.43 after 21 d fungus growth but no changes between these two fungi under the same Al3+ addition. Fungal Al3+ immobilization showed a three-stage trend with initially a rapid rate followed a relatively slower rate until reaching equilibrium. The pseudo-second order model was the best (R2 = 0.98 and 0.99 for L. deliciosus 2 and P. tinctorius 715) to fit the experimentally observed data among the three models. Compared to P. tinctorius 715, L. deliciosus 2 also had greater intercept value, cation exchange capacity (CEC), and extracellular Al3+ proportion in fungal mycelia. Additionally, bio-concentration on Al3+, active site numbers for Al3+, boundary layer thickness, CEC, and immobilization on the cell wall in fungal mycelia were involved in ECM fungal Al-tolerance. These results show that both ECM fungi are Al-tolerant while L. deliciosus 2 is a promising indigenous ECM isolate with higher Al-tolerance in Southwest China, and they can be hence applied to the afforestation and ecological restoration in acidic soil.

Interactions of microplastics and cadmium on plant growth and arbuscular mycorrhizal fungal communities in an agricultural soil.

Microplastics (MPs) as emerging contaminants have attracted attention worldwide, but little is known on their interactions with metallic contaminants in soil-plant systems. Here, we investigated the interactions between MPs, i.e., polyethylene (PE) and polylactic acid (PLA), and cadmium (Cd) on plant performance and arbuscular mycorrhizal fungal community in an agricultural soil. PE showed no noticeable phytotoxicity, while 10% PLA decreased maize biomass and chlorophyll content in leaves. A significant interaction on root biomass occurred between PE and Cd, but not between PLA and Cd. Both PE and PLA caused increase in soil pH and DTPA-extractable Cd concentrations, but no alterations in Cd accumulation in plant tissues. Different numbers of endemic and total OTUs were observed in various treatments. The relative abundance of arbuscular mycorrhizal fungi (AMF) genera highly varied with MPs and Cd. MPs altered AMF community structure and diversity, depending on their type and dose. Coexisting Cd produced slight but significant interactions with MPs on the dominant AMF genera. Overall, plant growth and AMF community varied with MPs type and dose, Cd, and their interactions, and the high dose of PLA produced stronger phytotoxicity. In conclusion, coexisting MPs and Cd can jointly drive shifts in plant performance and root symbiosis, thereby posing additional risks for agroecosystems and soil biodiversity.

Strategy for phytomanagement in an area affected by iron ore dam rupture: A study case in Minas Gerais State, Brazil.

In 2015, the Fundão tailing dam collapsed over the district of Bento Rodrigues (Mariana, Minas Gerais, Brazil) causing deaths, hundreds of homeless families and incalculable environmental degradation. Environmentally, economically and socially sustainable strategies are needed for the recovery of the affected areas. The main goal of this study was to evaluate the development, biomass production and toxic mineral elements absorption of three species of aromatic grasses (Chrysopogon zizanioides, Cymbopogon citratus and Cymbopogon winterianus). These three species were planted on polypropylene pots filled with the iron ore tailings collected from the topsoil of the Bento Rodrigues district. The pots were fertilized with increasing doses of organic compost associated with mycorrhizae as a phytomanagement strategy. A 4 × 2 factorial scheme was used. The seedlings were fertilized with four doses of organic compost, with or without mycorrhizae. At the highest dose of the organic compost (2 kg.plant-1), the total dry matter (dry matter of the aerial part + dry matter of the roots) for C. zizanioides was 4.5 times higher than the control (tailing only). For C. winterianus and C. Citratus was 3.8 and 2.8 times higher than the control, respectively. Inoculation with mycorrhizae improved biomass production, especially in C. zizanioides. The Fe and Mn levels found in the aerial part of the plants fertilized with organic compost were lower than those just growing on the iron ore tailings for the three species, which suggest that the organic matter apparently helped the plants in the exclusion of the hazardous substances and therefore increased the tolerance to these adverse environmental conditions. C. zizanioides, associated with organic matter and mycorrhizae, would be the recommended species. The proposed phytomanagement strategy can have a significant contribution to the gradual recovery of the affected area and also serve as a source of income for the local population.

The impact of nanoparticles zero-valent iron (nZVI) and rhizosphere microorganisms on the phytoremediation ability of white willow and its response.

Soil contaminated with heavy metals (HMs) is a serious problem throughout the world that threatens all living organisms in the soil. Therefore, large-scale remediation is necessary. This study investigated a new combination of remediation techniques on heavy metal contaminated soil, phytoremediation, and soil amendment with nano-sized zero-valent iron (nZVI) and rhizosphere microorganisms. White willow (Salix alba L.) was grown for 160 days in pots containing Pb, Cu, and Cd and amended with 0, 150, and 300 (mg kg-1) of nZVI and rhizosphere microorganisms, including the arbuscular mycorrhizal fungus (AMF), Rhizophagus irregularis, and the plant growth promoting rhizobacteria (PGPR), Pseudomonas fluorescens. The results showed that inoculation with PGPR and AMF, particularly dual inoculation, improved plant growth as well as the physiological and biochemical parameters of white willow, and increased the bioconcentration factor (BCF) of Pb, Cu, and Cd. The low dose of nZVI significantly increased the root length and the leaf area of the seedlings and increased the BCF of Cd. In contrast, the high dose of nZVI had negative effects on the seedlings growth and the BCF of Pb and Cu, about - 32% and - 63%, respectively. Our results demonstrate that nZVI at low doses can improve plant performance in a phytoremediation context and that the use of beneficial rhizosphere microorganisms can minimize nZVI stress in plants and make them less susceptible to stress even under high dose conditions.

Arbuscular mycorrhizal fungi possess a CLAVATA3/embryo surrounding region-related gene that positively regulates symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is a beneficial association established between land plants and the members of a subphylum of fungi, the Glomeromycotina. How the two symbiotic partners regulate their association is still enigmatic. Secreted fungal peptides are candidates for regulating this interaction. We searched for fungal peptides with similarities with known plant signalling peptides. We identified CLAVATA (CLV)/EMBRYO SURROUNDING REGION (ESR)-RELATED PROTEIN (CLE) genes in phylogenetically distant AM fungi: four Rhizophagus species and one Gigaspora species. These CLE genes encode a signal peptide for secretion and the conserved CLE C-terminal motif. They seem to be absent in the other fungal clades. Rhizophagus irregularis and Gigaspora rosea CLE genes (RiCLE1 and GrCLE1) are transcriptionally induced in symbiotic vs asymbiotic conditions. Exogenous application of synthetic RiCLE1 peptide on Medicago truncatula affects root architecture, by slowing the apical growth of primary roots and stimulating the formation of lateral roots. In addition, pretreatment of seedlings with RiCLE1 peptide stimulates mycorrhization. Our findings demonstrate for the first time that in addition to plants and nematodes, AM fungi also possess CLE genes. These results pave the way for deciphering new mechanisms by which AM fungi modulate plant cellular responses during the establishment of AM symbiosis.

Exposure to ionizing radiation affects the growth of ectomycorrhizal fungi and induces increased melanin production and increased capacities of reactive oxygen species scavenging enzymes.

Ectomycorrhizal (EM) fungi form symbioses with dominant tree families in boreal, temperate and tropical ecosystems and are important drivers of ecosystem function. EM fungal hyphae extend over a large area making them susceptible to enhanced radiation levels from naturally occurring or anthropogenically originating radioisotopes in the rhizosphere. In this study, the in-vitro effects of ionizing radiation on the growth and biomass of EM fungi Suillus luteus, S. bovinus and Rhizopogon luteolus were investigated. EM fungal cultures were exposed to gamma radiation from a 137Cs source for 137 h in darkness at 21 °C at dose rates of 404, 108.5 and 54.9 mGy h-1 resulting in total absorbed doses of 55.21, 14.82 and 7.50 Gy respectively. Cultures grown in the dark at 21 °C but not exposed to the 137Cs source served as the control. Our results show that EM fungi vary in their sensitivity to ionizing radiation. EM fungi used in this study produced melanin and reactive oxygen species scavenging enzymes such as catalase and superoxide dismutase as a response to ionizing radiation.

Cadmium and arsenic responses in the ectomycorrhizal fungus Laccaria bicolor: glutathione metabolism and its role in metal(loid) homeostasis.

Ectomycorrhizal fungi play an important role in protecting their host plant from metal(loid) stresses by synthesizing various thiol rich compounds like metallothioneins and glutathione. We investigated the effect of cadmium (Cd) and arsenic (As) stress with a specific interest on glutathione (GSH) in the ectomycorrhizal fungus Laccaria bicolor. The total GSH levels inside the cell were significantly increased with increase in external metal(loid) stress. An analysis of the transcript levels of genes responsible for GSH synthesis, γ-glutamylcysteine synthetase (Lbγ-GCS) and glutathione synthetase (LbGS), using qPCR revealed that expression of both genes increased as a function of external metal(loid) concentration. The enzyme activity of both Lbγ-GCS and LbGS were increased with increase in external Cd and As concentration. Further, the functional role of Lbγ-GCS and LbGS genes in response to Cd and As stress was studied using their respective yeast mutant strains gsh1 Δ and gsh2 Δ . The mutant strains successfully expressed the two genes resulting in wild-type phenotype restoration of Cd and As tolerance. From these results, it was concluded that GSH act as a core component in the mycorrhizal defence system under Cd and As stress for metal(loid) homeostasis and detoxification.

Sympatric diploid and tetraploid cytotypes of Centaurea stoebe s.l. do not differ in arbuscular mycorrhizal communities and mycorrhizal growth response.

PREMISE OF THE STUDY: Genome duplication is associated with multiple changes at different levels, including interactions with pollinators and herbivores. Yet little is known whether polyploidy may also shape belowground interactions. METHODS: To elucidate potential ploidy-specific interactions with arbuscular mycorrhizal fungi (AMF), we compared mycorrhizal colonization and assembly of AMF communities in roots of diploid and tetraploid Centaurea stoebe s.l. (Asteraceae) co-occurring in a Central European population. In a follow-up greenhouse experiment, we tested inter-cytotype differences in mycorrhizal growth response by combining ploidy, substrate, and inoculation with native AMF in a full-factorial design. KEY RESULTS: All sampled plants were highly colonized by AMF, with the Glomeraceae predominating. AMF-community composition revealed by 454-pyrosequencing reflected the spatial distribution of the hosts, but not their ploidy level or soil characteristics. In the greenhouse experiment, the tetraploids produced more shoot biomass than the diploids did when grown in a more fertile substrate, while no inter-cytotype differences were found in a less fertile substrate. AMF inoculation significantly reduced plant growth and improved P uptake, but its effects did not differ between the cytotypes. CONCLUSIONS: The results do not support our hypotheses that the cytotype structure in a mixed-ploidy population of C. stoebe is mirrored in AMF-community composition and that ploidy-specific fungal communities contribute to cytotype co-existence. Causes and implications of the observed negative growth response to AMF are discussed.

Arbuscular mycorrhizal fungi associated with the babassu palm (Attalea speciosa) in the eastern periphery of Amazonia, Brazil / Fungos micorrízicos arbusculares associados com a palmeira babaçu (Attalea speciosa) na periferia leste da Amazônia, Brasil

Babassu, Attalea speciosa (Arecaceae) is a ruderal palm native to Amazonia, which turned dominant in frequently burned lands throughout the arc of deforestation and other degraded lands, in extreme cases attaining complete dominance. This study investigated arbuscular mycorrhizal fungi (AMF) as one possible explanation for the outstanding ecological success of this exceptional palm. We explored the relationships between the babassu palm and native arbuscular mycorrhizal fungi and babassu effects on the AMF richness and mycorrhizal inoculum potential (MIP) in the eastern periphery of Amazonia. For this purpose, we sampled topsoil (0-20 cm) at the onset of the rainy season from a 5-year-old secondary forest regrowth (SEC) area with three levels of babassu dominance (sites with 10, 50 and 70% babassu biomass shares), and at three distances (0, 2.5 and 4 m) from isolated babassu patches within a degraded pasture (PAS), both with five replications per treatment. Glomerospore density varied from 100 to 302 per gram of soil, 56% higher in SEC than PAS. We identified a total of 16 AMF species, with dominance of Acaulospora (six species) followed by Glomus (three species). AMF richness increased with babassu dominance in SEC sites, and reduced with distance from babassu patches within the PAS. The colonization rate of babassu roots was higher in SEC than in PAS, whereas MIP was similar in both areas and without treatment differences. Our study points to strong mycorrhizal association of the babassu palm as a potential mechanism for its outstanding ecological success in degraded lands.

Babaçu, Attalea speciosa (Arecaceae) é uma palmeira ruderal nativa da Amazônia, dominante em terras frequentemente queimadas ao longo do arco de desmatamento e outras áreas degradadas, em casos extremos atingindo domínio completo. Este estudo investigou os fungos micorrízicos arbusculares (FMA) como possível explicação do sucesso ecológico desta palmeira. Nós exploramos as relações entre o babaçu e glomerosporos, efeitos do babaçu na riqueza destes fungos e o potencial do inóculo micorrízico (PIM) na periferia oriental da Amazônia. Amostras de solo (0-20 cm) foram coletadas no início da estação chuvosa em uma área de floresta secundária (SEC) de cinco anos de idade e três níveis de dominância do babaçu (10, 50 e 70% de biomassa de babaçu) e a três distâncias (0; 2,5 e 4 m) de ilhas de babaçu isoladas em uma pastagem degradada (PAS), ambas com cinco repetições por tratamento. A densidade de esporos de FMA variou de 100 a 302 por grama de solo, sendo 56% maior em SEC do que em PAS. Dezesseis espécies de FMA foram identificadas, com predominância de Acaulospora (seis espécies) seguidos do gênero Glomus (três espécies). A riqueza destes fungos aumentou com o domínio da palmeira em SEC e reduziu com a distância das ilhas de babaçu em PAS. A taxa de colonização das raízes de babaçu foi superior nas áreas de SEC enquanto o PIM não apresentou diferenças entre os tratamentos. Nosso estudo aponta a uma forte associação micorrhízica da palmeira babaçu, um possível mecanismo central no seu sucesso ecológico em áreas degradadas.

Ecotoxicity evaluation and human risk assessment of an agricultural polluted soil.

The present study aims to evaluate the nature and level of chemical pollution as well as the potential toxicity and ecotoxicity of an agricultural soil irrigated by the water of Litani River. Our findings showed that the soil was mainly contaminated by alkanes (hentriacontane, octadecane, hexadecane) and metal trace elements (nickel, vanadium, chromium, and manganese). Soil organic extracts showed high cytotoxicity against human hepatic (HepG2) and bronchial epithelial cells (Beas-2B). Soil ecotoxicity was revealed by seed germination inhibition of several plant species (wheat, clover, alfalfa, tall fescue, and ryegrass) ranging from 7 to 30% on the polluted soil compared to non-polluted one. In addition, significant decreases in telluric microbial biomasses (bacterial and fungal biomasses), quantified by phospholipid fatty acids (PLFA) analysis were observed in polluted soil compared to non-contaminated soils. The density of the arbuscular mycorrhizal fungal (AMF) spores isolated from the polluted soil was about 316 spores/100 g. Three main AMF species were identified as Funelliformis mosseae, Septoglomus constrictum, and Claroideoglomus lamellosum. Moreover, 16 indigenous plant species were inventoried with Silybum marianum L. as the dominant one. Plant biodiversity indices (Shannon, Simpson, Menhinick, and Margaleff) were lower than those found in other contaminated soils. Finally, it was found that all the present plant species on this polluted site were mycorrhized, suggesting a possible protection of these plants against encountered pollutants, and the possibility to use AMF-assisted phytoremediation to clean-up such a site.

Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi.

High-through-put (HTP) screening for functional arbuscular mycorrhizal fungi (AMF)-associations is challenging because roots must be excavated and colonization evaluated by transcript analysis or microscopy. Here we show that specific leaf-metabolites provide broadly applicable accurate proxies of these associations, suitable for HTP-screens. With a combination of untargeted and targeted metabolomics, we show that shoot accumulations of hydroxy- and carboxyblumenol C-glucosides mirror root AMF-colonization in Nicotiana attenuata plants. Genetic/pharmacologic manipulations indicate that these AMF-indicative foliar blumenols are synthesized and transported from roots to shoots. These blumenol-derived foliar markers, found in many di- and monocotyledonous crop and model plants (Solanum lycopersicum, Solanum tuberosum, Hordeum vulgare, Triticum aestivum, Medicago truncatula and Brachypodium distachyon), are not restricted to particular plant-AMF interactions, and are shown to be applicable for field-based QTL mapping of AMF-related genes.

Diversity and growth-effects of ectomycorrhizal fungi of a Nothofagus pumilio forest in the Andes of Southern Chile / (Diversidad y efectos de crecimiento de hongos ectomicorrízicos en un bosque de Nothofagus pumilio en los andes del sur de Chile

Chilean temperate rainforests have unique climatic, edaphic and biotic conditions, constituting pre-industrial blueprint ecosystems. Mycorrhizal associations play a central role in the biogeochemical processes of these ecosystems´ functioning. Baseline forest ecology studies are necessary in order to better understand diversity patterns, specifically regarding mycorrhizal symbiosis. Therefore, here we describe the vegetation characteristics and the mycorrhizal relationships of vascular plants in a Nothofagus pumilio forest. We also describe, via morphological methods, the ectomycorrhizal diversity present in this forest. Additionally, we determined whether ectomycorrhizal inoculation confers positive growth effects on N. pumilio seedlings. We found that from 46 vascular plant species identified in this study, 42 (91%) were mycorrhizal and of these 33 (72%) were associated with arbuscular mycorrhizae (AM), two (the dominant trees N. pumilio and N. dombeyi) were forming ectomycorrhizae (EM), five were associated with ericoid mycorrhizae, two with orchid mycorrhizae, and four were nonmycorrhizal. Additionally, 26 EM species were detected of which 15 belong to Cortinarius. Finally, there were clear differences in the growth of N. pumilio seedlings inoculated with the ectomycorrhizal fungus Laccaria laccata compared to noninoculated plants. We suggest that mycorrhizal fungi play a key role in seedling colonization of harsh environments such as the Andean treeline.

Los bosques templados lluviosos de Chile tienen condiciones climáticas, edáficas y bióticas únicas, constituyendo ecosistemas preindustriales modelo. Las asociaciones micorrízicas juegan un rol central en los procesos biogeoquímicos del funcionamiento de estos ecosistemas. Por lo tanto, en este estudio describimos las características de la vegetación y las relaciones micorrízicas de las plantas vasculares de un bosque de Nothofagus pumilio. También describimos, vía métodos morfológicos, la diversidad ectomicorrízica presente en este bosque. Adicionalmente, determinamos si inoculaciones ectomicorrízicas confieren efectos de crecimiento positivos a semilleros de N. pumilio. Encontramos que, de 46 especies de plantas vasculares identificadas en este estudio, 42 (91%) son micorrízicas, y de estas, 33 (72%) están formando micorrizas arbusculares (AM), dos (los árboles dominantes N. pumilio y N. dombeyi) están asociados con ectomicorrizas (EM), cinco están asociadas con micorrizas ericoides, dos con micorrizas orquioides, y cuatro fueron nomicorrizadas. Adicionalmente, 26 especies de EM fueron detectadas, de las cuales 15 pertenecen a Cortinarius. Finalmente, hubo claras diferencias en el crecimiento de los semilleros de N. pumilio inoculados con el hongo ectomicorrízico Laccaria laccata, comparados a plantas no inoculadas. Sugerimos que los hongos micorrízicos juegan un rol clave en la colonización de ambientes severos por juveniles, como en el límite altitudinal andino.

Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii).

Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their effects on Tuber borchii ("bianchetto" truffle) mycelium growth and morphology, they were tested on human K562 cell line and Lentinula edodes pure culture and shown to be non-toxic. Using these nanoparticles as iron supplement, the truffles showed extremely efficient iron uptake of over 300 times that of a commercial product. This avoided morphological changes in T. borchii due to lack of iron during growth and, with optimum nanoparticle dosage, increased growth without cell wall disruption or alteration of protoplasmatic hyphal content, the nuclei, mitochondria, and rough endoplasmic reticula being preserved. No significant modifications in gene expression were observed. These advantages derive from the completely different mechanism of iron delivery to mycelia compared to commercial iron supplements. The present data, in fact, show the nanoparticles attached to the cell wall, then penetrating it non-destructively without damage to cell membrane, mitochondria, chromatin, or ribosome. Low dosage significantly improved mycelium growth, without affecting hyphal morphology. Increases in hyphal diameter and septal distance indicated a healthier state of the mycelia compared to those grown in the absence of iron or with a commercial iron supplement. These positive effects were confirmed by measuring fungal biomass as mycelium dry weight, total protein, and ergosterol content. This "green" method for biogenerating iron exopolysaccharide nanoparticles offers many advantages, including significant economic savings, without toxic effects on the ectomycorrhizal fungus, opening the possibility of using them as iron supplements in truffle plantations.

Regulatory networks underlying mycorrhizal development delineated by genome-wide expression profiling and functional analysis of the transcription factor repertoire of the plant symbiotic fungus Laccaria bicolor.

BACKGROUND: Ectomycorrhizal (ECM) fungi develop a mutualistic symbiotic interaction with the roots of their host plants. During this process, they undergo a series of developmental transitions from the running hyphae in the rhizosphere to the coenocytic hyphae forming finger-like structures within the root apoplastic space. These transitions, which involve profound, symbiosis-associated metabolic changes, also entail a substantial transcriptome reprogramming with coordinated waves of differentially expressed genes. To date, little is known about the key transcriptional regulators driving these changes, and the aim of the present study was to delineate and functionally characterize the transcription factor (TF) repertoire of the model ECM fungus Laccaria bicolor. RESULTS: We curated the L. bicolor gene models coding for transcription factors and assessed their expression and regulation in Poplar and Douglas fir ectomycorrhizae. We identified 285 TFs, 191 of which share a significant similarity with known transcriptional regulators. Expression profiling of the corresponding transcripts identified TF-encoding fungal genes differentially expressed in the ECM root tips of both host plants. The L. bicolor core set of differentially expressed TFs consists of 12 and 22 genes that are, respectively, upregulated and downregulated in symbiotic tissues. These TFs resemble known fungal regulators involved in the control of fungal invasive growth, fungal cell wall integrity, carbon and nitrogen metabolism, invasive stress response and fruiting-body development. However, this core set of mycorrhiza-regulated TFs seems to be characteristic of L. bicolor and our data suggest that each mycorrhizal fungus has evolved its own set of ECM development regulators. A subset of the above TFs was functionally validated with the use of a heterologous, transcription activation assay in yeast, which also allowed the identification of previously unknown, transcriptionally active yet secreted polypeptides designated as Secreted Transcriptional Activator Proteins (STAPs). CONCLUSIONS: Transcriptional regulators required for ECM symbiosis development in L. bicolor have been uncovered and classified through genome-wide analysis. This study also identifies the STAPs as a new class of potential ECM effectors, highly expressed in mycorrhizae, which may be involved in the control of the symbiotic root transcriptome.

In vitro micropropagation and mycorrhizal treatment influences the polyphenols content profile of globe artichoke under field conditions.

The commercial importance of plant tissue culture has grown in recent years, reflecting its application to vegetative propagation, disease elimination, plant improvement and the production of polyphenols. The level of polyphenols present in plant tissue is influenced by crop genotype, the growing environment, the crop management regime and the post-harvest processing practice. Globe artichoke is a significant component of the Mediterranean Basin agricultural economy, and is rich in polyphenols (phenolic acids and flavones). Most commercially grown plants are derived via vegetative propagation, with its attendant risk of pathogen build-up. Here, a comparison was drawn between the polyphenol profiles of conventionally propagated and micropropagated/mycorrhized globe artichoke plants. Micropropagation/mycorrhization appeared to deliver a higher content of caffeoylquinic acids. The accumulation of these compounds, along with luteolin and its derivatives, was not season-dependent. Luteolin aglycone was accumulated preferentially in the conventionally propagated plants. Overall, it appeared that micropropagation/mycorrhization enhanced the accumulation of polyphenols.

Crescimento de Ruta graveolens l. Inoculada com micorrizas sob doses de fósforo / Growth of Ruta graveolens l. Inoculated with arbuscular mycorrhizal fungi under levels of phosphorus / Crecimiento del Ruta graveolens l. Inoculada con micorrizas bajo dosis de fósforo

Os fungos micorrízicos arbusculares (FMAs) são micro-organismos que fazem simbiose com raízes da maioria das plantas. O objetivo deste trabalho foi avaliar a influência dos FMAs Rhizophagus clarus e Claroideoglomus etunicatum em substrato sob baixa e ou alta dose de fósforo (P), na produção de biomassa e na acumulação de P na arruda (Ruta graveolens L.). O experimento foi conduzido no laboratório de botânica da Universidade Paranaense - UNIPAR, município de Umuarama - PR. O delineamento experimental foi inteiramente ao acaso, em fatorial 3x2, sendo os fatores: FMAs (sem FMA, com R. clarus e ou com C. etunicatum); duas doses de P (20 e 200 mg kg-1) com 8 repetições por tratamento, num total de 48 unidades experimentais (vasos de 3 L). Avaliou-se a produção de massa seca das raízes (MSR), massa seca da parte aérea (MSPA), massa seca total (MST) e o acúmulo de P na parte aérea da planta (PPA). A produção de MSR, MSPA e MST pela planta não foram significativamente afetadas apenas pela adição de P ao substrato, porém sim, pela inoculação com o FMA C. etunicatum, sob baixo e ou alto P. O acúmulo de P na parte aérea da planta foi aumentado significativamente no tratamento sem a inoculação com FMAs. Conclui-se que a inoculação com C. etunicatum é indicada para aumentar a produtividade da arruda.(AU)

Arbuscular mycorrhizal fungi (AMF) are microorganisms that present symbiosis with the roots of most plants. The purpose of this work was to evaluate the influence of AMF Rhizophagus clarus and Claroideoglomus etunicatum on low and high doses of phosphorus (P) on plant biomass and P accumulation in rue (Ruta graveolens L.). The experiment was performed in the botanical laboratory at Universidade Paranaense ­ UNIPAR in the city of Umuarama, in the state of Paraná, Brazil. It used a completely randomized 3x2 experimental design, being: AMFs (without AMF, with R. clarus and/or with C. etunicatum); two levels of P (20 and 200 mg kg-1) with 8 repetitions per treatment, totaling 48 experimental units (3 L pots). Root dry matter (RDM), shoot dry matter (SDM), total dry matter (TDM) and P accumulation in the aerial part of the plant were evaluated. The production of RDM, SDM and TDM by the plant was not significantly affected by the addition of P to the substrate, but by the inoculation with AMF C. etunicatum under low and/or high P. The accumulation of P in the aerial part of the plant significantly increased in the treatment without AMF inoculation. It was concluded that inoculation with C. etunicatum is indicated to increase the productivity of rue.(AU)

Los hongos micorrícicos arbusculares (HMA) son microorganismos que forman simbiosis con las raíces de la mayoría de las plantas. El objetivo de este estudio fue evaluar la influencia del HMA Rhizophagus clarus y Claroideoglomus etunicatum en substrato so baja y/o alta dosis de fósforo (P), en la producción de biomasa y en la acumulación de P en la ruda (Ruta graveolens L.). El experimento se llevó a cabo en el laboratorio de botánica de la Universidad Paranaense - Unipar, municipio de Umuarama - PR. El diseño experimental fue completamente al azar, en un 3x2 factorial, con los factores: HMA (Sin HMA, con R. clarus y C. etunicatum); dos dosis de P (20 y 200 mg kg-1) con 8 repeticiones por tratamiento de un total de 48 unidades experimentales (vasos de 3 L). Se evaluó la producción de materia seca de las raíces (MSR), materia seca de la parte aérea (MSPA), materia seca total (MST) y la acumulación de P en la parte aérea de la planta (PPA). La producción de MSR, MSPA y MST por la planta no se vieron afectados significativamente sólo mediante la adición de P al sustrato, sino por inoculación con HMA C. etunicatum so bajo y/o alto P. El acúmulo de P en parte aérea de la planta se incrementó significativamente en el tratamiento sin inoculación con HMA. Se concluye que la inoculación con C. etunicatum está indicada para aumentar la productividad de la ruda.(AU)

Gene expression analyses in tomato near isogenic lines provide evidence for ethylene and abscisic acid biosynthesis fine-tuning during arbuscular mycorrhiza development.

Plant responses to the environment and microorganisms, including arbuscular mycorrhizal fungi, involve complex hormonal interactions. It is known that abscisic acid (ABA) and ethylene may be involved in the regulation of arbuscular mycorrhiza (AM) and that part of the detrimental effects of ABA deficiency in plants is due to ethylene overproduction. In this study, we aimed to determine whether the low susceptibility to mycorrhizal colonization in ABA-deficient mutants is due to high levels of ethylene and whether AM development is associated with changes in the steady-state levels of transcripts of genes involved in the biosynthesis of ethylene and ABA. For that, tomato (Solanum lycopersicum) ethylene overproducer epinastic (epi) mutant and the ABA-deficient notabilis (not) and sitiens (sit) mutants, in the same Micro-Tom (MT) genetic background, were inoculated with Rhizophagus clarus, and treated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). The development of AM, as well as the steady-state levels of transcripts involved in ethylene (LeACS2, LeACO1 and LeACO4) and ABA (LeNCED) biosynthesis, was determined. The intraradical colonization in epi, not and sit mutants was significantly reduced compared to MT. The epi mutant completely restored the mycorrhizal colonization to the levels of MT with the application of 10 µM of AVG, probably due to the inhibition of the ACC synthase gene expression. The steady-state levels of LeACS2 and LeACO4 transcripts were induced in mycorrhizal roots of MT, whereas the steady-state levels of LeACO1 and LeACO4 transcripts were significantly induced in sit, and the steady-state levels of LeNCED transcripts were significantly induced in all genotypes and in mycorrhizal roots of epi mutants treated with AVG. The reduced mycorrhizal colonization in sit mutants seems not to be limited by ethylene production via ACC oxidase regulation. Both ethylene overproduction and ABA deficiency impaired AM fungal colonization in tomato roots, indicating that, besides hormonal interactions, a fine-tuning of each hormone level is required for AM development.

Production of native arbuscular mycorrhizal fungi inoculum under different environmental conditions

Abstract In order to obtain an arbuscular mycorrhizal fungi (AMF) native inoculum from Sierra de Moa and determine the most appropriate conditions for its big scale production, four light and temperature combinations were tested in three plant species (Calophyllum antillanum, Talipariti elatum and Paspalum notatum). Growth and development parameters, as well as the mycorrhizal functioning of the seedlings were evaluated. The natural light treatment under high temperatures (L-H) was the most suitable for the growth and development of the three plant species, showing the highest total biomass values, mainly of root, and a positive root-shoot ratio balance. This treatment also promoted higher values of root mycorrhizal colonization, external mycelium and AMF spore density. A total of 38 AMF species were identified among the plants and environmental conditions tested. Archaeospora sp.1, Glomus sp.5, Glomus brohultii and G. glomerulatum were observed in all the treatments. The L-H condition can be recommended for native inoculum production, as it promotes a better expression of the AM symbiosis and an elevated production of mycorrhizal propagules.

Positive Gene Regulation by a Natural Protective miRNA Enables Arbuscular Mycorrhizal Symbiosis.

Arbuscular mycorrhizal (AM) symbiosis associates most plants with fungi of the phylum Glomeromycota. The fungus penetrates into roots and forms within cortical cell branched structures called arbuscules for nutrient exchange. We discovered that miR171b has a mismatched cleavage site and is unable to downregulate the miR171 family target gene, LOM1 (LOST MERISTEMS 1). This mismatched cleavage site is conserved among plants that establish AM symbiosis, but not in non-mycotrophic plants. Unlike other members of the miR171 family, miR171b stimulates AM symbiosis and is expressed specifically in root cells that contain arbuscules. MiR171b protects LOM1 from negative regulation by other miR171 family members. These findings uncover a unique mechanism of positive post-transcriptional regulation of gene expression by miRNAs and demonstrate its relevance for the establishment of AM symbiosis.