Structural analyzes suggest that MiSSP13 and MiSSP16.5 may act as proteases inhibitors during ectomycorrhiza establishment in Laccaria bicolor.

•The signaling process during mycorrhiza establishment involves intense molecular communication between symbionts. It has been suggested that a group of protein effectors, the so-called MiSSPs, plays a broader function in the symbiosis metabolism, however, many of these remain uncharacterized structurally and functionally. •Herein we used three-dimensional protein structure modeling methods, ligand analysis, and molecular docking to structurally characterize and describe two protein effectors, MiSSP13 and MiSSP16.5, with enhanced expression during the mycorrhizal process in Laccaria bicolor. •MiSSP13 and MiSSP16.5 show structural homology with the cysteine and aspartate protease inhibitor, cocaprin (CCP1). Through structural analysis, it was observed that MiSSP13 and MiSSP16.5 have an active site similar to that observed in CCP1. The protein-protein docking data showed that MiSSP13 and MiSSP16.5 interact with the papain and pepsin proteases at sites that are near to where CCP1 interacts with these same targets, suggesting a function as inhibitor of cysteine and aspartate proteases. The interaction of MiSSP13 with papain and MiSSP16.5 with pepsin was stronger than the interaction of CCP1 with these proteases, suggesting that the MiSSPs had a greater activity in inhibiting these classes of proteases. Based on the data supplied, a model is proposed for the function of MiSSPs 13 and 16.5 during the symbiosis establishment. Our findings, while derived from in silico analyses, enable us formulate intriguing hypothesis on the function of MiSSPs in ectomycorrhization, which will require experimental validation.

Ectomycorrhizal fungi, two species of Laccaria, differentially block the migration and accumulation of cadmium and copper in Pinus densiflora.

The root tips of host plant species can establish ectomycorrhizae with their fungal partners, thereby altering the responses of the host plants to heavy metal (HM) toxicity. Here, two species of Laccaria, L. bicolor and L. japonica, were investigated in symbiosis with Pinus densiflora to study their potential for promotion of phytoremediation of HM-contaminated soils in pot experiments. The results showed that L. japonica had significantly higher dry biomass than L. bicolor in mycelia grown on modified Melin-Norkrans medium containing elevated levels of cadmium (Cd) or copper (Cu). Meanwhile, the accumulations of Cd or Cu in L. bicolor mycelia were much higher than that in L. japonica at the same level of Cd or Cu. Therefore, L. japonica displayed a stronger tolerance to HM toxicity than L. bicolor in situ. Compared with non-mycorrhizal P. densiflora seedlings, inoculation with two Laccaria species significantly increased the growth of P. densiflora seedlings in absence or presence of HM. The mantle of host roots blocked the uptake and migration of HM, which led to the decrease of Cd and Cu accumulation in the P. densiflora shoots and roots, except for the root Cd accumulation of L. bicolor-mycorrhizal plants when 25 mg kg-1 Cd exposure. Furthermore, HM distribution in mycelia showed Cd and Cu are mainly retained in the cell walls of mycelia. These results provide strong evidence that the two species of Laccaria in this system may have different strategies to assist host tree against HM toxicity.

Mycorrhiza-induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity.

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.

Identification and characterization of eight metallothionein genes involved in heavy metal tolerance from the ectomycorrhizal fungus Laccaria bicolor.

Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification. The increasing numbers of available genomic sequences of ectomycorrhizal (ECM) fungi enable deeper insights into the characteristics of MT genes in these fungi that form the most important symbiosis with the host trees in forest ecosystems. The aim of this study was to establish a comprehensive, genome-wide inventory of MT genes from the ECM fungus Laccaria bicolor. Eight MT genes in L. bicolor were cloned, and the expression patterns of their transcripts at various developmental stages based on expressed sequence tag (EST) counts were analyzed. The expression levels of four MTs were significantly increased during symbiosis stages. Quantitative real-time PCR (qRT-PCR) analysis revealed that transcripts of LbMT1 were dominant in free-living mycelia and strongly induced by excessive copper (Cu), cadmium (Cd), and hydrogen peroxide (H2O2). To determine whether these eight MTs functioned as metal chelators, we expressed them in the Cu- and Cd-sensitive yeast mutants, cup1∆ and yap1∆, respectively. All LbMT proteins provided similar levels of Cu(II) or Cd(II) tolerance, but did not affect by H2O2. Our findings provide novel data on the evolution and diversification of fungal MT gene duplicates, a valuable resource for understanding the vast array of biological processes in which these proteins are involved.

Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.

l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding α-keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His-hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His-frLAAO1 from Fibroporia radiculosa and 9His-laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N-glycosylated. We generated mutants of 9His-hcLAAO4 lacking N-glycosylation sites to further understand the effects of N-glycosylation. All four predicted N-glycosylation sites were glycosylated in 9His-hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His-hcLAAO4 and variants without one or all N-glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N-glycans. This was caused by the absence of a hypermannosylated N-glycan on asparagine residue N54. The lack of one or all of the other N-glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N-glycosylation during biosynthesis.

A Viable New Strategy for the Discovery of Peptide Proteolytic Cleavage Products in Plant-Microbe Interactions.

Small peptides that are proteolytic cleavage products (PCPs) of less than 100 amino acids are emerging as key signaling molecules that mediate cell-to-cell communication and biological processes that occur between and within plants, fungi, and bacteria. Yet, the discovery and characterization of these molecules is largely overlooked. Today, selective enrichment and subsequent characterization by mass spectrometry-based sequencing offers the greatest potential for their comprehensive characterization, however qualitative and quantitative performance metrics are rarely captured. Herein, we addressed this need by benchmarking the performance of an enrichment strategy, optimized specifically for small PCPs, using state-of-the-art de novo-assisted peptide sequencing. As a case study, we implemented this approach to identify PCPs from different root and foliar tissues of the hybrid poplar Populus × canescens 717-1B4 in interaction with the ectomycorrhizal basidiomycete Laccaria bicolor. In total, we identified 1,660 and 2,870 Populus and L. bicolor unique PCPs, respectively. Qualitative results supported the identification of well-known PCPs, like the mature form of the photosystem II complex 5-kDa protein (approximately 3 kDa). A total of 157 PCPs were determined to be significantly more abundant in root tips with established ectomycorrhiza when compared with root tips without established ectomycorrhiza and extramatrical mycelium of L. bicolor. These PCPs mapped to 64 Populus proteins and 69 L. bicolor proteins in our database, with several of them previously implicated in biologically relevant associations between plant and fungus.

Laccaria bicolor Mobilizes both Labile Aluminum and Inorganic Phosphate in Rhizosphere Soil of Pinus massoniana Seedlings Field Grown in a Yellow Acidic Soil.

Plant growth is often limited by highly activated aluminum (Al) and low available phosphorus (P) in acidic soil. Ectomycorrhizal (ECM) fungi can improve their host plants' Al tolerance by increasing P availability while decreasing Al activity in vitro or in hydroponic or sand culture systems. However, the effect of ECM fungi on inorganic P (IP) and labile Al in acidic soil in the field, particularly in conjunction with Al treatment, remains poorly understood. The present study aimed to determine the influence of ECM fungal association on the mobilization of IP and labile Al in rhizosphere soil of host plants grown in the field with external Al treatment and the underlying nutritional mechanism in plant Al tolerance. To do so, 4-week-old Pinus massoniana seedlings were inoculated with three ECM isolates (Laccaria bicolor 270, L. bicolor S238A, and L. bicolor S238N) and grown in a Haplic Alisol field with or without Al treatment for 12 weeks. Results showed that L. bicolor association enhanced the available P depletion and facilitated the mobilization of IP and labile Al, in turn improving the capacity of host plant to use Al-bound P, Ca-bound P, and occluded P, particularly when P. massoniana seedlings were inoculated with L. bicolor S238A. Inoculation with L. bicolor isolates also enhanced the solubility of labile Al and facilitated the conversion of acid-soluble Al into exchangeable Al. Our findings suggested that ECM inoculation could enhance plant Al tolerance in the field by mobilizing IP to improve the P bioavailability but not by decreasing Al activity.IMPORTANCE Here, we reveal the underlying nutritional mechanism in plant Al tolerance conferred by ectomycorrhizal (ECM)-fungus inoculation in the field and report the screening of a promising ECM isolate to assist phytoremediation and afforestation using Pinus massoniana in acidic soil in southern China. This study advances our understanding of the contribution of ECM fungi to plant-ECM-fungus symbiosis and highlights the vital role of ECM-fungus inoculation in plant Al tolerance. In addition, the results described in the present study confirm the importance of carrying out studies in the field rather than only in vitro studies. Our findings strengthen our understanding of the role of ECM-fungus association in detecting, utilizing, and transporting unavailable nutrients in the soil to enhance host plant growth and adaptability in response to adverse habitats.

Physicochemical and in vitro cytotoxic properties of chitosan from mushroom species (Boletus bovinus and Laccaria laccata).

Chitosan samples from two mushroom species (Boletus bovinus, Laccaria laccata) were obtained and characterized by viscosimetry, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), elemental analyses (EA), nuclear magnetic resonance spectroscopy (13C NMR), X-ray diffraction (XRD) and thermogravimetric (TGA) analyses. Properties of the fungal chitosan samples were compared to commercial low-molecular weight chitosan, crustacean chitosan (Cervimunida johni) and chitosan obtained from an insect (Hilobius abietis). Additionally, the cytotoxic properties of chitosan in vitro on cancerous hepatoma and non-cancerous ovary cells cultivated on films with different chitosan concentrations was evaluated. As a conclusion, this study clearly revealed that low-molecular weight chitosan films and solutions with high degree of deacetylation can act cytotoxically on both tumor MH-22A and normal CHO cells in vitro. Consequently, this work may be useful for further investigations of natural anticancer products in medical areas.

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.

DeltaMS: a tool to track isotopologues in GC- and LC-MS data.

INTRODUCTION: Stable isotopic labeling experiments are powerful tools to study metabolic pathways, to follow tracers and fluxes in biotic and abiotic transformations and to elucidate molecules involved in metal complexing. OBJECTIVE: To introduce a software tool for the identification of isotopologues from mass spectrometry data. METHODS: DeltaMS relies on XCMS peak detection and X13CMS isotopologue grouping and then analyses data for specific isotope ratios and the relative error of these ratios. It provides pipelines for recognition of isotope patterns in three experiment types commonly used in isotopic labeling studies: (1) search for isotope signatures with a specific mass shift and intensity ratio in one sample set, (2) analyze two sample sets for a specific mass shift and, optionally, the isotope ratio, whereby one sample set is isotope-labeled, and one is not, (3) analyze isotope-guided perturbation experiments with a setup described in X13CMS. RESULTS: To illustrate the versatility of DeltaMS, we analyze data sets from case-studies that commonly pose challenges in evaluation of natural isotopes or isotopic signatures in labeling experiment. In these examples, the untargeted detection of sulfur, bromine and artificial metal isotopic patterns is enabled by the automated search for specific isotopes or isotope signatures. CONCLUSION: DeltaMS provides a platform for the identification of (pre-defined) isotopologues in MS data from single samples or comparative metabolomics data sets.

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.

Differential expression of metallothioneins in response to heavy metals and their involvement in metal tolerance in the symbiotic basidiomycete Laccaria bicolor.

Cysteine-rich peptides such as metallothioneins (MTs) are involved in metal homeostasis and detoxification in many eukaryotes. We report the characterization and expression of two MT genes, LbMT1 and LbMT2 from the ectomycorrhizal fungus Laccaria bicolor under metal stress conditions. LbMT1 and LbMT2 differ with respect to the length of the encoded peptides (58 versus 37 aa, respectively) and also by their expression patterns in response to metals. The expression levels of both LbMT1 and LbMT2 increased as a function of increased external Cu concentration, the expression levels for LbMT2 were always significantly higher compared with those of LbMT1. Only LbMT1, but not LbMT2, responded to Cd supply in the range of 25-100 µM while Zn did not affect the transcription of either LbMT1 or LbMT2. Both genes also responded to oxidative stress, but to a lesser extent compared to their responses to either Cu or Cd stress. Heterologous complementation assays in metal-sensitive yeast mutants indicated that both LbMT1 and LbMT2 encode peptides capable of conferring higher tolerance to both Cu and Cd. The present study identified LbMTs as potential determinants of the response of this mycorrhizal fungus to Cu and Cd stress.

Ethylene and jasmonic acid act as negative modulators during mutualistic symbiosis between Laccaria bicolor and Populus roots.

The plant hormones ethylene, jasmonic acid and salicylic acid have interconnecting roles during the response of plant tissues to mutualistic and pathogenic symbionts. We used morphological studies of transgenic- or hormone-treated Populus roots as well as whole-genome oligoarrays to examine how these hormones affect root colonization by the mutualistic ectomycorrhizal fungus Laccaria bicolor S238N. We found that genes regulated by ethylene, jasmonic acid and salicylic acid were regulated in the late stages of the interaction between L. bicolor and poplar. Both ethylene and jasmonic acid treatments were found to impede fungal colonization of roots, and this effect was correlated to an increase in the expression of certain transcription factors (e.g. ETHYLENE RESPONSE FACTOR1) and a decrease in the expression of genes associated with microbial perception and cell wall modification. Further, we found that ethylene and jasmonic acid showed extensive transcriptional cross-talk, cross-talk that was opposed by salicylic acid signaling. We conclude that ethylene and jasmonic acid pathways are induced late in the colonization of root tissues in order to limit fungal growth within roots. This induction is probably an adaptive response by the plant such that its growth and vigor are not compromised by the fungus.

Iron ore weathering potentials of ectomycorrhizal plants.

Plants in association with soil microorganisms play an important role in mineral weathering. Studies have shown that plants in symbiosis with ectomycorrhizal (ECM) fungi have the potential to increase the uptake of mineral-derived nutrients. However, it is usually difficult to study many of the different factors that influence ectomycorrhizal weathering in a single experiment. In the present study, we carried out a pot experiment where Pinus patula seedlings were grown with or without ECM fungi in the presence of iron ore minerals. The ECM fungi used included Pisolithus tinctorius, Paxillus involutus, Laccaria bicolor and Suillus tomentosus. After 24 weeks, harvesting of the plants was carried out. The concentration of organic acids released into the soil, as well as potassium and phosphorus released from the iron ore were measured. The results suggest that different roles of ectomycorrhizal fungi in mineral weathering such as nutrient absorption and transfer, improving the health of plants and ensuring nutrient circulation in the ecosystem, are species specific, and both mycorrhizal roots and non-mycorrhizal roots can participate in the weathering process of iron ore minerals.

A secreted effector protein of Laccaria bicolor is required for symbiosis development.

Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues and thus can be considered a mutualism effector.

Biological activity of the Agrobacterium rhizogenes-derived trolC gene of Nicotiana tabacum and its functional relation to other plast genes.

Agrobacterium rhizogenes induces hairy roots through the activity of three essential T-DNA genes, rolA, rolB, and rolC, whereas the orf13 gene acts as an accessory root-inducing gene. rolB, rolC, and orf13 belong to the highly diverged plast gene family with remotely related representatives in the endomycorrhizal basidiomycete Laccaria bicolor. Nicotiana glauca and N. tabacum contain A. rhizogenes-derived T-DNAs with active plast genes. Here, we report on the properties of a rolC homolog in N. tabacum, trolC. Dexamethasone-inducible trolC and A4-rolC genes from A. rhizogenes A4 induce comparable, strong growth effects affecting all parts of the plants. Several have not been described earlier and were found to be very similar to the effects of the distantly related plast gene 6b. They include leaf chlorosis and starch accumulation, enations, increase of sucrose-dependent leaf disk expansion, growth of isolated roots on low-sucrose media, and stimulation of sucrose uptake by small root fragments. Collectively, our findings indicate that enhancement of sucrose uptake plays an important role in generating the complex 6b and rolC phenotypes and might be an ancestral property of the plast genes.

A new isoprenyl phenyl ether riboside from the culture of basidiomycete Laccaria amethystea.

A new isoprenyl phenyl ether riboside, 3-(3-methylbut-2-enyloxy)-4-O-alpha-D-ribofuranose benzoic acid methyl ester (1), was isolated from the culture of basidiomycete Laccaria amethystea. The structure of 1 was elucidated on the basis of extensive spectroscopic analysis.

Determinación de la actividad inmunomoduladora de los basidiomicetos comestibles: Cantharellus lateritius singer (Berk) Singer, Armillariella olymyces (Pers:Letell Sing y Clem, Laccaria amethystina Cooke, Lactarius deliciosus (L. ex Fr) S.F Gray y Pleurotus ostreatus (Jacquim ex Fries) Kummer

Fue evaluada la actividad de extractos acuosos y etánolicos de cinco especies de basidiomicetos comestibles; Armillariella polymyces (Silip en Q´ eqchi´), Cantharellus lateritius (Anacate), Laccaria amethystina (sombrerito, sombrero de Xara, monja), Lactarius deliciosus (Shara amarilla, amacaria, cabeza de xara) Pleurotus ostreatus (Hongo ostra, hongo blanco), sobre la proliferación de linfocitos y la activación del sistema de complemento. El efecto sobre la linfoproliferación, fue medido evaluando la viabilidad celular de linfocitos humanos que fueron enfrentados a diferentes concentraciones de extracto acuoso y etanólico de cada basidiomiceto. Los resultados obtenidos mostraron actividad inhibitoria inespecífica (ya que no se encontró efecto de dosis-respuesta)...

Caracterización del cultivo miceliar de Laccaria bicolor P. D Orton Agaricales en medio MMN con diferentes pH / Characterizacion of mycelial culture of Laccaria bicolor in MM medium with different

Se caracterizó el crecimiento miceliar in vitro de una cepa nativa de Laccaria bicolor (196,1997), recolectada en Huehuetenango, Guatemala, en medio Melin-Norkrans Modificado (MMN) con cuatro niveles de pH; 4.5, 5.5, 6.5 y 7.0, incubando las placas durante 40 días a 26 centígrados. Se determinó que el mayor diámetro de las colonias se obtuvo en el pH 7.0. La morfología de las colonias varió según los tratamientos; con pH 4.5 y 5.5 la cepa produjo acúmulo hifales, mientras que con pH 6.5 y 7.0 las colonias fueron lisas y fibrilosas. Microscópicamente se observaron hifas de 2.0-4.0...