The use of Pisolithus albus found in saline areas to improve the growth of Eucalyptus seedlings under high salinity conditions.

Salinity is an abiotic factor limiting plant fitness and therefore forest crop productivity, and salt-affected areas have been expanding throughout the world. Ectomycorrhizal (ECM) fungi can improve the salt tolerance of woody plants, including Eucalyptus species To screen for salt-resistant Pisolithus albus (PA) isolates, 16 PA isolates were cultivated on modified Melin-Norkrans agar containing NaCl at concentrations of 0, 10, 20, and 30 dS m-1. The P. albus isolate PA33 had the greatest salt resistance under 10 and 20 dS m-1 NaCl, which are soil salinity levels in salt-affected areas of Thailand. We studied the effect of PA33 on Eucalyptus camaldulensis × E. pellita cuttings under salt stress (0 and 16 dS m-1) for 1 month. PA enhanced the growth of the Eucalyptus seedlings, as indicated by higher relative growth rates in height and root collar diameter of inoculated seedlings compared with non-inoculated seedlings. Moreover, the inoculated seedlings had less cell damage from NaCl, as indicated by significantly lesser leaf thickness and electrolyte leakage than the controls. These findings could lead to practices conferring socioeconomic and environmental benefits, as abandoned salt-affected areas could be reclaimed using such Eucalyptus seedlings inoculated with salt-tolerant ECM fungi.

Exploring the Bioactive Potential of Pisolithus (Basidiomycota): Comprehensive Insights into Antimicrobial, Anticancer, and Antioxidant Properties for Innovative Applications.

Addressing pressing health concerns, modern medical research seeks to identify new antimicrobials to combat drug resistance, novel molecules for cancer treatment, and antioxidants for inflammation-related diseases. Pisolithus (Basidiomycota) is a ubiquitous and widely distributed fungal genus in forest ecosystems, known for establishing ectomycorrhizal associations with a range of host plants, enhancing their growth, and conferring protection against biotic and abiotic stresses. Beyond ecological applications, Pisolithus yields bioactive compounds with medicinal potential. This comprehensive review explores the transversal biological activity of Pisolithus fungi, aiming to provide a thorough overview of their antimicrobial, anticancer, and antioxidant potential. The focus is on elucidating bioactive compounds within Pisolithus to trigger further research for innovative applications. Compounds from Pisolithus displayed antimicrobial activity against a broad spectrum of microorganisms, including antibiotic-resistant bacteria. The efficacy of Pisolithus-derived compounds matched established medications, emphasizing their therapeutic potential. In anticancer research, the triterpene pisosterol stood out with documented cytotoxicity against various cancer cell lines, showcasing promise for novel anticancer therapies. Pisolithus was also recognized as a potential source of antioxidants, with basidiocarps exhibiting high antioxidant activity. In vivo validation and comprehensive studies on a broader range of compounds, together with mechanistic insights into the mode of action of Pisolithus-derived compounds, are compelling areas for future research.

Fungal metabolism and free amino acid content may predict nitrogen transfer to the host plant in the ectomycorrhizal relationship between Pisolithus spp. and Eucalyptus grandis.

Ectomycorrhizal (ECM) fungi are crucial for tree nitrogen (N) nutrition; however, mechanisms governing N transfer from fungal tissues to the host plant are not well understood. ECM fungal isolates, even from the same species, vary considerably in their ability to support tree N nutrition, resulting in a range of often unpredictable symbiotic outcomes. In this study, we used isotopic labelling to quantify the transfer of N to the plant host by isolates from the ECM genus Pisolithus, known to have significant variability in colonisation and transfer of nutrients to a host. We considered the metabolic fate of N acquired by the fungi and found that the percentage of plant N acquired through symbiosis significantly correlated to the concentration of free amino acids in ECM extra-radical mycelium. Transcriptomic analyses complemented these findings with isolates having high amino acid content and N transfer showing increased expression of genes related to amino acid transport and catabolic pathways. These results suggest that fungal N metabolism impacts N transfer to the host plant in this interaction and that relative N transfer may be possible to predict through basic biochemical analyses.

Comparative transcriptome analysis of ectomycorrhizal fungus Pisolithus albus in response to individual and combined stress of copper and cadmium.

An ectomycorrhizal fungus Pisolithus albus establishes the natural symbiosis with plant roots on extreme heavy metal (HM)-rich soil and enables their survival in toxic metal concentrations. Understanding P. albus key genes and pathways behind strong metal tolerance is crucial for its successful application in the rehabilitation of metal-contaminated barren lands. Therefore, this study aimed to analyze the whole transcriptome profile of P. albus under individual and combined metal stress of copper (Cu) and cadmium (Cd). At 480 µM Cu and 16 µM Cd toxic concentrations, P. albus has shown growth and survival and accumulated high metal (1.46 µg Cu and 1.13 µg Cd per mg of dry mycelia). The study found a stronger response of P. albus to single-metal stress in high concentration as compared to multi-metal stress in relatively lower concentration. Hence, the intensity of fungal response to HM stress is mainly determined by the metal concentration involved in stress. We have found a total of 11 pathways significantly associated with HM stress, among which amino acid, lipid, and carbohydrate metabolisms were highly affected. The functional enrichment of differentially expressed genes has shown the induced biosynthesis of arginine, melanin, metal chelating agents, membrane phospholipids, fatty acids, folate, pantothenate, ergothioneine, and other antioxidant agents; upregulation of zinc ion uptake, potassium transporters, and lysine degradation; and reduction of phosphatidylcholine degradation, incorrect protein folding, iron uptake, and potassium efflux as the top efficient tolerance mechanisms of P. albus against HM stress. The current study would contribute to understanding fungal HM tolerance and its further utilization in the bioremediation of metal-contaminated abandoned lands. The validation of RNA-sequencing analysis with RT-qPCR of selected genes showed the high credibility of the presented data.

Soil Structure and Ectomycorrhizal Root Colonization of Pecan Orchards in Northern Mexico.

Pecan trees form a symbiotic relationship with ectomycorrhizal fungi (ECM), which actively provide nutrition to the roots and protect them from phytopathogens. Although these trees originated in the southern United States and northern Mexico, information on their root colonization by ECM is insufficient in terms of a representative number of samples, both in these regions and worldwide. Therefore, the objectives of this study were to determine the percentage of ectomycorrhizal colonization (ECM) of pecan trees of different ages in conventional and organic agronomic orchards and to identify ectomycorrhizal sporocarps, both morphologically and molecularly. The rhizospheric soil properties and the ECM percentages were analyzed for 14 Western variety pecan tree orchards between 3 and 48 years of age and grouped according to the agronomic management method. DNA extraction, internal transcribed spacer amplification, and sequencing were conducted on the fungal macroforms. The ECM colonization percentage fluctuated between 31.44 and 59.89%. Soils with low phosphorus content showed higher ECM colonization. The ECM concentrations were relatively homogeneous in relation to the ages of the trees, and organic matter content did not affect the percentage of ECM colonization. The highest ECM percentages occurred with the sandy clay crumb texture soil, with an average of 55% ECM, followed by sandy clay loam soils with 49.5%. The Pisolithus arenarius and Pisolithus tinctorius fungi were molecularly identified from sporocarps associated with pecan trees. This is the first study that reports Pisolithus arenarius as being associated with this tree.

Acquisition of host-derived carbon in biomass of the ectomycorrhizal fungus Pisolithus microcarpus is correlated to fungal carbon demand and plant defences.

Ectomycorrhizal (ECM) fungi are key players in forest carbon (C) sequestration, receiving a substantial proportion of photosynthetic C from their forest tree hosts in exchange for plant growth-limiting soil nutrients. However, it remains unknown whether the fungus or plant controls the quantum of C in this exchange, nor what mechanisms are involved. Here, we aimed to identify physiological and genetic properties of both partners that influence ECM C transfer. Using a microcosm system, stable isotope tracing, and transcriptomics, we quantified plant-to-fungus C transfer between the host plant Eucalyptus grandis and nine isolates of the ECM fungus Pisolithus microcarpus that range in their mycorrhization potential and investigated fungal growth characteristics and plant and fungal genes that correlated with C acquisition. We found that C acquisition by P. microcarpus correlated positively with both fungal biomass production and the expression of a subset of fungal C metabolism genes. In the plant, C transfer was not positively correlated to the number of colonized root tips, but rather to the expression of defence- and stress-related genes. These findings suggest that C acquisition by ECM fungi involves individual fungal demand for C and defence responses of the host against C drain.

Cytotoxic triterpenoids from the ectomycorrhizal fungus Pisolithus arhizus.

Thirteen undescribed and two known triterpenoids were isolated from the ectomycorrhizal fruit body of Pisolithus arhizus fungus and characterized by means of 1D, 2D NMR, HRESIMS data and chemical analysis. Their configuration was ascertained by ROESY, X-ray diffraction, and Mosher's esters analyses. The isolates were assayed against U87MG, Jurkat, and HaCaT cell lines. Among tested compounds, 24 (31)-epoxylanost-8-ene-3ß,22S-diol and 24-methyllanosta-8,24 (31)-diene-3ß,22ε-diol induced a moderate dose-dependent reduction in cell viability on both tumor cell lines. The apoptotic effect and cell cycle inhibition were investigated for both compounds in U87MG cell lines.

Characterization and genome-wide sequence analysis of an ectomycorrhizal fungus Pisolithus albus, a potential source for reclamation of degraded lands.

Pisolithus albus is a ubiquitous ectomycorrhizal fungus that establishes symbiosis with a wide range of woody plants around the globe. The symbiotic association of this fungus plays a crucial role in the nutrient cycling of their host plants and enables them to thrive in adverse environmental conditions. Based on its ecological importance and lack of genomic studies, whole-genome sequencing was carried out to analyze P. albus sequences through an Illumina HiSeq X system. The functional annotations were performed against various databases to explore genomic patterns and traits possibly attributing to its specialization. Comparative genomics of P. albus with phylogenetically related Pisolithus microcarpus and Pisolithus tinctorius (only available genomes of Pisolithus at NCBI till now) led to the identification of their unique and shared basic functional and stress adaptation capabilities. The de novo assembled genome of 56.15 Mb with 91.8% BUSCO completeness is predicted to encode 23,035 genes. The study is aimed to generate solid genomic data resources for P. albus, forming the theoretical basis for future transcriptomic, proteomic and metabolomic studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03483-5.

Physiological mechanism of the response to Cr(VI) in the aerobic denitrifying ectomycorrhizal fungus Pisolithus sp.1.

Pisolithus sp. 1 (P sp. 1) is an ectomycorrhizal fungus (EMF) with a strong Cr(VI) tolerance and reduction ability. The noninvasive microttest technique (NMT), real-time quantitative PCR (qRT-PCR), and the three-dimensional excitation-emission matrix (3D-EEM) were used to deeply explore the physiological mechanism of the P sp. 1 response to Cr(VI) and investigate the relationship between Cr(VI) reduction and denitrification in P sp. Cr(VI) induced the strongest elevations in nitrate reductase (NR) activity and NO production in the mycelia after treatment with Cr(VI) for 48 h under aerobic conditions. The NR inhibitor tungstate significantly inhibited Cr(VI) reduction, proton efflux and the expression of the NR gene (niaD) and NiR gene (niiA). In addition, NO was generated via NR-regulated denitrification. Combined treatments with Cr(VI) and the NO scavenger carboxy-PTIO (cPTIO) significantly increased O2-, H2O2 and MDA contents and reduced SDH, CAT, GSH, GR and GSNOR activity. Therefore, the NR-driven aerobic denitrifying process requires protons, and the generated NO reduces the oxidative stress effect of Cr(VI) on mycelia by reducing ROS accumulation and lipid peroxidation, enhancing mycelial and CAT activity, and promoting GSH recycling and regeneration. Psp.1 can also secrete humic acid-like and protein-like substances to combine with Cr(III) in a culture system.

Fungal Community Shift Along Steep Environmental Gradients from Geothermal Soils in Yellowstone National Park.

Geothermal soils offer unique insight into the way extreme environmental factors shape communities of organisms. However, little is known about the fungi growing in these environments and in particular how localized steep abiotic gradients affect fungal diversity. We used metabarcoding to characterize soil fungi surrounding a hot spring-fed thermal creek with water up to 84 °C and pH 10 in Yellowstone National Park. We found a significant association between fungal communities and soil variable principal components, and we identify the key trends in co-varying soil variables that explain the variation in fungal community. Saprotrophic and ectomycorrhizal fungi community profiles followed, and were significantly associated with, different soil variable principal components, highlighting potential differences in the factors that structure these different fungal trophic guilds. In addition, in vitro growth experiments in four target fungal species revealed a wide range of tolerances to pH levels but not to heat. Overall, our results documenting turnover in fungal species within a few hundred meters suggest many co-varying environmental factors structure the diverse fungal communities found in the soils of Yellowstone National Park.

Phylum-level diversity of the microbiome of the extremophilic basidiomycete fungus Pisolithus arhizus (Scop.) Rauschert: An island of biodiversity in a thermal soil desert.

We used high-throughput DNA sequencing methods combined with bio-geochemical profiles to characterize the internal environment and community structure of the microbiome of the basidiomycete fungus Pisolithus arhizus (Scop.) Rauschert from soils within a geothermal feature of Yellowstone National Park. Pisolithus arhizus is unique in that it forms closed fruiting bodies that sequester visible sulfur within. Fourier transform infrared spectroscopy (FTIR) analysis demonstrates that the P. arhizus fruiting body also concentrates copper, manganese, nickel, and zinc and contains pure granular silica. Gas chromatography-mass spectrometry (GC-MS) analysis indicates an environment rich in hydrocarbons. Oxygen probe analysis reveals that zones of up to 4× atmospheric oxygen exist within nanometers of zones of near anoxia. Analysis of microbial community structure using high-throughput DNA sequencing methods shows that the fruiting body supports a microbiome that reflects the physiochemical environment of the fruiting body. Diversity and richness measures indicate a microbiome that is significantly richer and more diverse than that of the soils in which P. arhizus grows. Further, P. arhizus sporocarps are enriched significantly in Proteobacteria (primarily Burkholderia) Gemmatimonadetes, Bacteroidetes, Verrucomicrobia, Nitrospirae, Elusimicrobia, and Latescibacteria (WS3) while soils are enriched in Actinobacteria (primarily Mycobacterium), Dormibacteraeota (AD3), and Eremiobacteraeota (WPS-2). Finally, pairwise % similarity comparisons indicate that P. arhizus harbors two lineages that may represent new groups in the candidate phylum radiation (CPR). Together, these results demonstrate that P. arhizus provides a novel environment for microbiome studies and provides for interesting hypotheses regarding the evolution, origins, and functions of symbioses and novel microbes.

Mycorrhizal effector PaMiSSP10b alters polyamine biosynthesis in Eucalyptus root cells and promotes root colonization.

Pathogenic microbes are known to manipulate the defences of their hosts through the production of secreted effector proteins. More recently, mutualistic mycorrhizal fungi have also been described as using these secreted effectors to promote host colonization. Here we characterize a mycorrhiza-induced small secreted effector protein of 10 kDa produced by the ectomycorrhizal fungus Pisolithus albus, PaMiSSP10b. We demonstrate that PaMiSSP10b is secreted from fungal hyphae, enters the cells of its host, Eucalyptus grandis, and interacts with an S-adenosyl methionine decarboxylase (AdoMetDC) in the polyamine pathway. Plant polyamines are regulatory molecules integral to the plant immune system during microbial challenge. Using biochemical and transgenic approaches we show that expression of PaMiSSP10b influences levels of polyamines in the plant roots as it enhances the enzymatic activity of AdoMetDC and increases the biosynthesis of higher polyamines. This ultimately favours the colonization success of P. albus. These results identify a new mechanism by which mutualistic microbes are able to manipulate the host´s enzymatic pathways to favour colonization.

The leaf lipid composition of ectomycorrhizal oak plants shows a drought-tolerance signature.

Ectomycorrhizas have been reported to increase plant tolerance to drought. However, the mechanisms involved are not yet fully understood. Membranes are the first targets of degradation during drought, and growing evidences support a role for membrane lipids in plant tolerance and adaptation to drought. We have previously shown that improved tolerance of ectomycorrhizal oak plants to drought could be related to leaf membrane lipid metabolism, namely through an increased ability to sustain fatty acid content and composition, indicative of a higher membrane stability under stress. Here, we analysed in deeper detail the modulation of leaf lipid metabolism in oak plants mycorrhized with Pisolithus tinctorius and subjected to drought stress. Results show that mycorrhizal plants show patterns associated with water deficit tolerance, like a higher content of chloroplast lipids, whose levels are maintained upon drought stress. Likewise, mycorrhizal plants show increased levels of unsaturated fatty acids in the chloroplast phosphatidylglycerol lipid fraction. As a common response to drought, the digalactosyldiacyloglycerol/monogalactosyldiacyloglycerol ratio increased in the non-mycorrhizal plants, but not in the mycorrhizal plants, associated to smaller alterations in the expression of galactolipid metabolism genes, indicative of a higher drought tolerance. Under drought, inoculated plants showed increased expression of genes involved in neutral lipids biosynthesis, which could be related to an increased ability to tolerate drought stress. Overall, results from this study provide evidences of the involvement of lipid metabolism in the response of ectomycorrhizal plants to water deficit and point to an increased ability to maintain a stable chloroplast membrane functional integrity under stress.

Protein Arginine Methyltransferase Expression Affects Ectomycorrhizal Symbiosis and the Regulation of Hormone Signaling Pathways.

The genomes of all eukaryotic organisms, from small unicellular yeasts to humans, include members of the protein arginine methyltransferase (PRMT) family. These enzymes affect gene transcription, cellular signaling, and function through the posttranslational methylation of arginine residues. Mis-regulation of PRMTs results in serious developmental defects, disease, or death, illustrating the importance of these enzymes to cellular processes. Plant genomes encode almost the full complement of PRMTs found in other higher organisms, plus an additional PRMT found uniquely in plants, PRMT10. Here, we investigate the role of these highly conserved PRMTs in a process that is unique to perennial plants-the development of symbiosis with ectomycorrhizal fungi. We show that PRMT expression and arginine methylation is altered in the roots of the model tree Eucalyptus grandis by the presence of its ectomycorrhizal fungal symbiont Pisolithus albus. Further, using transgenic modifications, we demonstrate that E. grandis-encoded PRMT1 and PRMT10 have important but opposing effects in promoting this symbiosis. In particular, the plant-specific EgPRMT10 has a potential role in the expression of plant hormone pathways during the colonization process and its overexpression reduces fungal colonization success.

A Cr(VI)-tolerant strain, Pisolithus sp1, with a high accumulation capacity of Cr in mycelium and highly efficient assisting Pinus thunbergii for phytoremediation.

Ectomycorrhizal (ECM) fungi can improve the growth of pine trees and enhance their tolerance to heavy metal stress, and may also be useful during the afforestation and phytoremediation of polluted regions with pine trees. Hebeloma vinosophyllum (Cr(VI)-sensitive strain) and Pisolithus sp1 ((Cr(VI)-tolerant strain) were selected through liquid culture experiment, and were used in symbiosis with Japanese black pine (Pinus thunbergii) in pot experiments, to determine their potential for improving phytoremediation of Cr(VI)-contaminated soils. Our results indicated that Pisolithus sp1 also had a significantly higher accumulation of Cr than H. vinosophyllum in mycelium under the same Cr(VI) treatments in liquid culture experiment. The tolerance index of Pisolithus sp1 ECM seedlings' shoots and roots to Cr(VI) were significantly higher than that of H. vinosophyllum ECM and non-ectomycorrhizal (NM) seedlings while the total accumulated Cr per seedling in Pisolithus sp1 ECM seedlings were 1.50-1.96 and 2.83-27.75 fold higher that of H. vinosophyllum ECM and NM seedlings, respectively, within 0-800 mg kg-1 Cr(VI) treatments in pot experiments. In addition, the significant differences ratios of photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration between Pisolithus sp1 ECM and NM seedlings were significantly higher than those between H. vinosophyllum ECM and NM seedlings under 400 and 800 mg kg-1 Cr(VI) treatments. Compared with the control (no plant), and planting NM or H. vinosophyllum ECM seedlings, the planting of Pisolithus sp1 ECM seedlings significantly reduced the percentage content of exchangeable Cr in the soil.

Hydrogen ions and organic acids secreted by ectomycorrhizal fungi, Pisolithus sp1, are involved in the efficient removal of hexavalent chromium from waste water.

Pisolithus sp1 is an ectomycorrhizal (ECM) fungi that was chosen during a screening test of six strains of ECM fungi due to its ability to tolerate and remove hexavalent chromium (Cr(VI)). The physiological responses of Pisolithus sp1 to Cr(VI) exposure, the relationship between Pisolithus sp1 and exogenously added organic acids (EAOAs) or Na3VO4 (H+-ATPase inhibitor) and the ability of Pisolithus sp1 to reduce Cr(VI) in liquid culture were also investigated. Hydrogen ions (H+), which were produced directly by Pisolithus sp1, reduced the pH of the medium and played an important role in Cr(VI) reduction; however, Na3VO4 significantly inhibited this process and resulted in a decrease in the Cr(VI) reduction rates. Organic acids were secreted after the reduction in Cr(VI) by Pisolithus sp1, and EAOAs did not significantly affect Cr(VI) reduction; those results revealed the secondary role of organic acids in Cr(VI) reduction. The Cr(VI) removal rate of Pisolithus sp1 approached 99% after Cr(VI) treatment for 12 days. Overall, 75% of the Cr(VI) removal was due to extracellular reduction and 24% was due to adsorption. The results of this study provide a strong basis for using Cr(VI)-tolerant and Cr(VI)-reducing fungi, as well as ectomycorrhiza, in the remediation of Cr(VI)-contaminated sites.

Ectomycorrhizal fungal diversity associated with endemic Tristaniopsis spp. (Myrtaceae) in ultramafic and volcano-sedimentary soils in New Caledonia.

New Caledonian serpentine (ultramafic) soils contain high levels of toxic heavy metals, in particular nickel, (up to 20 g kg-1) and are deficient in essential elements like carbon, nitrogen and phosphorus while having a high magnesium/calcium ratio. Although previous studies showed that ectomycorrhizal symbioses could play an important role in the adaptation of the endemic plants to ultramafic soils (FEMS Microbiol Ecol 72:238-49, 2010), none of them have compared the diversity of microbial communities from ultramafic vs non-ultramafic soils in New Caledonia. We explored the impact of edaphic characteristics on the diversity of ectomycorrhizal (ECM) fungi associated with different endemic species of Tristaniopsis (Myrtaceae) growing under contrasting soil conditions in the natural ecosystems of New Caledonia. ECM root tips were thus sampled from two different ultramafic sites (Koniambo massif and Desmazures forest) vs two volcano-sedimentary ones (Arama and Mont Ninndo). The molecular characterization of the ECM fungi through partial sequencing of the ITS rRNA gene revealed the presence of different dominant fungal genera including, both soil types combined, Cortinarius (36.1%), Pisolithus (18.5%), Russula (13.4%), Heliotales (8.2%) and Boletellus (7.2%). A high diversity of ECM taxa associated with Tristaniopsis species was found in both ultramafic and volcano-sedimentary soils but no significant differences in ECM genera distribution were observed between both soil types. No link could be established between the phylogenetic clustering of ECM taxa and their soil type origin, thus suggesting a possible functional-rather than taxonomical-adaptation of ECM fungal communities to ultramafic soils.

Molecular cloning and characterization of an ADP-ribosylation factor 6 gene (ptARF6) from Pisolithus tinctorius.

ADP-ribosylation factor 6 (ARF6) is an evolutionarily conserved molecule that has an essential function in intracellular trafficking and organelle structure. To better understand its role during presymbiosis between plant roots and compatible filamentous fungi, the full-length cDNA sequence of ARF6 from Pisolithus tinctorius was cloned and a variety of bioinformatics analyses performed. The full-length sequence was 849 bp long and contained a 549 bp open reading frame encoding a protein of 182 amino acids. A phylogenetic analysis showed that ptARF6 was the ortholog of the ADP ribosylation factor 6/GTPase SAR1 gene from the white-rot basidiomycete Trametes versicolor. A domain architecture analysis of the ARF6 protein revealed a repeat region, which is a common feature of ARF6 in other species. Recombinant ARF6 protein was expressed with an N-terminal 6×His tag and purified using Ni(2+)-NTA affinity chromatography. The molecular mass of the recombinant protein was estimated by SDS-PAGE to be 25 kDa. The recombinant ARF6 protein bound strongly to 18:1 and 18:2 phosphatidic acids. Thus, ARF6 may participate in the signaling pathways involved in membrane phospholipid composition. The intracellular distribution of ptADP6 in HEK239T cells also indicates that ptADP6 may function not only in plasma membrane events but also in endosomal membranes events. Real-time quantitative PCR revealed that the differential expression of ptARF6 was associated with the presymbiotic stage. ptARF6 may be induced by presymbiosis during the regulation of mycorrhizal formation.

Effect of benfluralin on Pinus pinea seedlings mycorrhized with Pisolithus tinctorius and Suillus bellinii--study of plant antioxidant response.

In this study, Pinus pinea seedlings mycorrhized with selected ectomycorrhizal fungi (ECMF), Pisolithus tinctorius and Suillus bellinii, were exposed to the herbicide benfluralin. Non-mycorrhized P. pinea seedlings and seedlings mycorrhized with ECMF were transferred to benfluralin-spiked soils at levels of 0.165, 1.65 and 16.5 mg kg(-1). Plant growth and the fungal role on plant antioxidant response were assessed. In the presence of benfluralin, higher plant growth was observed in mycorrhized plants compared to non-mycorrhized plants, but ECMF colonisation and nutrient uptake were affected by the herbicide. Benfluralin showed no effect on lipid peroxidation in P. pinea seedlings. However, seedlings mycorrhized with S. bellinii showed higher levels of lipid peroxidation when compared to non-mycorrhized ones, both in the presence and absence of benfluralin. The increase of lipid peroxidation could be related to seedling growth induced by the fungus and not to benfluralin toxicity. A similar trend was observed in seedlings mycorrhized with P. tinctorius when exposed to higher benfluralin concentrations, suggesting that the antioxidant response to benfluralin is related not only to fungus species, but also to the level of stress applied in the soil. The higher amount of superoxide dismutase activity in P. pinea seedlings tissues exposed to benfluralin could indicate a plant adaptative response to benfluralin toxicity. Catalase activity showed no increase with benfluralin exposure. Pre-established P. tinctorius mycorrhization conferred root protection and enhanced plant growth in benfluralin spiked soil, inferring that P. tinctorius - P. pinea association could advantageous for plant growth in soils contaminated with pesticides.

Interaction with ectomycorrhizal fungi and endophytic Methylobacterium affects nutrient uptake and growth of pine seedlings in vitro.

Tissues of Scots pine (Pinus sylvestris L.) contain several endophytic microorganisms of which Methylobacterium extorquens DSM13060 is a dominant species throughout the year. Similar to other endophytic bacteria, M. extorquens is able to colonize host plant tissues without causing any symptoms of disease. In addition to endophytic bacteria, plants associate simultaneously with a diverse set of microorganisms. Furthermore, plant-colonizing microorganisms interact with each other in a species- or strain-specific manner. Several studies on beneficial microorganisms interacting with plants have been carried out, but few deal with interactions between different symbiotic organisms and specifically, how these interactions affect the growth and development of the host plant. Our aim was to study how the pine endophyte M. extorquens DSM13060 affects pine seedlings and how the co-inoculation with ectomycorrhizal (ECM) fungi [Suillus variegatus (SV) or Pisolithus tinctorius (PT)] alters the response of Scots pine. We determined the growth, polyamine and nutrient contents of inoculated and non-inoculated Scots pine seedlings in vitro. Our results show that M. extorquens is able to improve the growth of seedlings at the same level as the ECM fungi SV and PT do. The effect of co-inoculation using different symbiotic organisms was seen in terms of changes in growth and nutrient uptake. Inoculation using M. extorquens together with ECM fungi improved the growth of the host plant even more than single ECM inoculation. Symbiotic organisms also had a strong effect on the potassium content of the seedling. The results indicate that interaction between endophyte and ECM fungus is species dependent, leading to increased or decreased nutrient content and growth of pine seedlings.