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.

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.

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.

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.

Co-metabolic degradation of mono-fluorophenols by the ectomycorrhizal fungi Pisolithus tinctorius.

The release of fluorinated organic compounds from fire retardants or agrochemical products may have a significant negative effect on soil ecosystems. In this study, the ability of Pisolithus tinctorius to tolerate and degrade mono-fluorophenols (FP) was assessed. In vitro studies showed fungal growth in the presence of 0.45mM of 2-FP and 3-FP, but not in the presence of 4-FP. P. tinctorius was able to degrade up to 79% and 92% of 1mM 2-FP and 3-FP, respectively, in glucose supplemented liquid medium, suggesting that 2- and 3-FP degradation occurred in co-metabolism with glucose consumption. 3-Fluorocatechol (FC) and 4-FC were identified as metabolic intermediates using HPLC and LC-MS. Liberation of fluoride was not detected suggesting that a fluorinated dead-end product was formed. In extracts of cells collected at the end of cultures supplemented with the mono-FPs, a metabolic intermediate compatible with a mass corresponding to a fluoromuconate compound, according to LC-MS data, was recovered. The results further suggest that ectomycorrhizal fungi may be able to degrade mono-FP in pure culture while using glucose as a carbon source, through a similar pathway as that found in bacteria. To our knowledge, this is the first time that degradation of mono-FPs by an ectomycorrhizal fungus is reported.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots / PtSRR1, um possível receptor simbiose-regulado de Pisolithus tinctorius é expresso nas primeiras horas de interação ectomicorrízica com raízes de Castanea sativa

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

PtSRR1 foi isolado preliminarmente de P. tinctorius nas primeiras horas da interação com raízes de C. sativa. Análises de QRT-PCR confirmaram sua expressão positiva (12 h) e seu peptídeo putativo indicou forte possibilidade para proteína transmembranar. Estes dados estimulam o estudo do PtSRR1 durante a formação de ectomicorrizas.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots.

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

Host plant stimulates hypaphorine accumulation in Pisolithus tinctorius hyphae during ectomycorrhizal infection while excreted fungal hypaphorine controls root hair development.

The hypaphorine concentration in Pisolithus tinctorius Coker & Couch hyphae colonizing Eucalyptus roots was 3 to 5 times higher than in adjacent parts of the fungal colony. This phenomenon, observed 24 h after inoculation, was also recorded in several-month-old, well-established ectomycorrhizas. Accumulation was controlled by specific root-derived diffusible molecules: it can be induced through a membrane, but not by non-host plants. In pure culture, high hypaphorine concentration was found only in the youngest mycelium, i.e. the outer 2 mm of the colony. Fungal hypaphorine had no IAA-like activity on Eucalyptus root development and therefore could not be considered as an auxin analogue; instead, a strong reduction of root hair elongation was recorded.

Cloning symbiosis-related cDNAs from eucalypt ectomycorrhiza by PCR-assisted differential screening.

As part of a project to identify symbiosis-related genes, we report here a simple differential screening procedure for isolating up- and down-regulated fungal transcripts from a cDNA library of the developing Eucalyptus globulus-Pisolithus tinctorius mycorrhiza. cDNA inserts of randomly selected λZAP plaques were amplified by PCR and separated by agarose gel electrophoresis. The PCR-amplified cDNA samples were then screened by Southern blotting, using radiolabelled-cDNA probes of high specific activity. We have applied this method to fungal transcripts that are differentially expressed in ectomycorrhizas during the early stages of development. We estimate that about 50 % of the fungal mRNA population is regulated by development of the symbiosis; several up- and down-regulated cDNAs have been isolated for further analysis.

Utilization of microbial siderophores by mycorrhizal and non-mycorrhizal pine roots.

In iron-deficient conditions, most bacteria and fungi are known to release siderophores, iron-chelating compounds. Most plants do not produce siderophores, but seem to use microbial siderophores as iron sources. Although ectomycorrhizal fungi have been found to release siderophores in pure culture, little research has addressed production by mycorrhizas and the consequences for plant iron nutrition. The objectives of this study were to determine the effect of an ectomycorrhizal fungus [Pisolithus tinctorius (Pers.) Coker and Couch] on the utilization of siderophore (ferrioxamine B) by slash pine (Pinus elliottii Engelm.) roots grown under iron-deficient or iron-sufficient conditions. Experiments were conducted with excised roots and whole seedlings. Uptake of 55 Fe from ferrioxamine B was lower by mycorrhizal roots than non-mycorrhizal roots. Growth under iron-deficient conditions had little effect on iron uptake by non-mycorrhizal roots but increased the uptake by mycorrhizal roots. Uptake of iron from a non-purified siderophore isolated from a pure culture of P. tinctorius was also lower by mycorrhizal roots. The uptake of iron was not dependent on the pH of the uptake solution. Differential responses could be attributed to different mechanisms of iron uptake between fungal cells and root cells. However, the higher iron content of mycorrhizal roots may indicate a negative feedback effect on uptake.

Effects of high soil moisture on formation of ectomycorrhizas and growth of karri (Eucalyptus diversicolor) seedlings inoculated with Descolea maculata, Pisolithus tinctorius and Laccaria laccata.

Seedlings of Eucalyptus diversicolor P. Muell. I noculated with the ectomycorrhizal fungi Descolea maculata Bougher (two isolates), Pisolithus tinctorius (Pers.) Coker & Couch and Laccaria laccata (Scop, ex Fr.) Berk. & Br. were raised under glasshouse conditions in a yellow sand at a gradient of four soil moisture levels ranging from above field capacity to near waterlogged. All fungi enhanced growth of seedlings above that of uninoculated seedlings, but in soils near saturation there was no response to inoculation. Reduced mycorrhizal formation in relation to increasing soil moisture occurred to various degrees for all fungi. This was particularly marked with Pisolithus tinctorius. In contrast, Laccaria laccata maintained a relatively high number of mycorrhizal roots at all moisture levels applied, except at the wettest soil treatment. An isolate of D. maculata from a swamp environment did not produce a greater number of mycorrhizal roots at high soil moisture than an isolate of this species from a forest environment.

Growth and phosphorus acquisition of karri (Eucalyptus diversicolor F. Muell.) seedlings inoculated with ectomycorrhizal fungi in relation to phosphorus supply.

Growth and phosphorus acquisition of pot-grown seedlings of karri (Eucalyptus diversicolor F. Muell.) were examined following inoculation with four ectomycorrhizal fungi -Descolea maculata Bougher (two isolates), Pisolithus tinctorius (Pers.) Coker & Couch, and Laccaria laccata (Scop, ex Fr.) Berk. & Br. Seedlings were raised in steam-sterilized sand to which 13 rates of phosphorus (0.100 mg P kg-1 soil) were applied. All fungi except P. tinctorius produced a plant growth response. L. laccata produced the largest growth response. Responses were greatest at low rates of application of P to soil. There was no effect of the fungi on growth at levels of P application above 28 mg P kg-1 soil. A threshold effect (no increase in growth with increasing additions of P) characteristic of non-mycorrhizal seedlings was eliminated by mycorrhizal infection. Mycorrhizal inoculation increased P content of plant tissues at sub-optimal levels of P supply. The effect of mycorrhizas on seedling P status diminished with increasing soil P. One isolate of D. maculata often had greater rates of P accumulation and produced higher concentrations of P in plant tissues than L. laccata, but did not produce greater plant biomass. Frequency of infection for all fungi was low in soils with no additional P, and greatest with the addition of 2 mg P kg-1 soil (L. laccata and D. maculata isolate A), or 4 mg P kg-1 soil (D. maculata isolate B). Infection was reduced with increasing soil P, and not evident at 36 mg P kg-1 soil or higher levels of soil P. L. laccata had higher infection frequency and mycorrhizal root length at all levels of soil P than the D. maculata isolates. Two fungi produced basidiomes. This occurred at levels of soil P application ranging from 4 to 28 mg P kg-1 soil for D. maculata (isolate B), and at 4 to 28 mg P kg-1 soil for L. laccata.

Ectomycorrhizal formation by micropropagated clones of Eucalyptus marginata inoculated with isolates of Pisolithus tinctorius.

Eucalyptus marginata Donn ex Sm. and Pisolithus tinctorius (Pers.) Cok and Couch were co-cultured to obtain ectomycorrhizal formation in vitro. One isolate of P. tinctorius formed mycorrhizas with aseptic seedlings of a juvenile clone derived from a 4-month-old seedling, and four clones derived from crowns of mature trees. A second P. tinctorius isolate formed mycorrhizas with only the clones from mature trees. Successful combinations resulted in formation of a mantle followed by a Hartig net and epidermal cell elongation. The fungal/seedlings or fungal/seedling clone combinations which did not produce ectomycorrhizal roots, were characterized by a mantle but lacked a Hartig net, and formed an abundance of polyphenols throughout the root. Genotype, maturity and fungal specificity are key factors influencing successful ectomycorrhizal formation on E. marginata by P. tinctorius in vitro.