Vegetative Mycelium Research Articles

Discovery of microRNA-like RNAs during early fruiting body development in the model mushroom Coprinopsis cinerea.

Coprinopsis cinerea is a model mushroom particularly suited for the study of fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) have been extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), one for Argonaute (AGO), one for AGO-like and one for quelling deficient-2 (QDE-2) proteins, were also identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to those in animals and plants. Taken together, our findings provided the first evidence for milRNAs in the model mushroom and their potential roles in regulating fruiting body development. New information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights for guiding further functional and evolutionary studies of miRNAs.

Pathogenicity of Ganoderma Species on Landscape Trees in the Southeastern United States.

The genus Ganoderma contains species that are associated with dead and declining host trees. Many species have been described as pathogens in literature, because anecdotally, the presence of fruiting bodies on living trees has been widely associated with a general decline in tree health. Few studies have investigated the pathogenicity of Ganoderma species on landscape trees in the southeastern U.S. Pathogenicity tests were used to determine the pathogenicity of G. curtisii, G. meredithiae, G. sessile, and G. zonatum on young, healthy landscape trees (Pinus elliottii var. elliottii, P. taeda, Quercus shumardii, Q. virginiana, and Butia odorata) common to the southeastern U.S. Inoculations were made by drilling into the sapwood of the lower bole and inserting wooden dowels that were infested with each Ganoderma species. In two field experiments, 11 to 12 months post inoculation, trees had no visual, external symptoms of disease. There were differences in the extent of internal xylem discoloration near the site of inoculation in comparison with the mock-inoculated control in experiment 1, but there were no differences relative to the control in experiment 2. In both experiments, G. sessile was the only species that was successfully reisolated from the pine and oak hosts. Although disease symptoms were not obvious, the reisolation of G. sessile outside the inoculation point was a significant finding, and suggests that this species was capable of infecting healthy sapwood. G. sessile constitutively produces chlamydospores within its vegetative mycelium, which may contribute to its persistence in the discolored sapwood. These data suggest that the Ganoderma species tested, following trunk wounding, are not pathogens of young, actively growing landscape trees that only possess sapwood. The establishment of these fungi using alternative infection courts (e.g., roots) and their interactions in older living trees (e.g., trees with heartwood) needs investigation to better understand their effects on tree health.

Production of Prodiginines Is Part of a Programmed Cell Death Process in Streptomyces coelicolor.

Actinobacteria are prolific producers of antitumor antibiotics with antiproliferative activity, but why these bacteria synthetize metabolites with this bioactivity has so far remained a mystery. In this work we raised the hypothesis that under certain circumstances, production of antiproliferative agents could be part of a genetically programmed death of the producing organism. While programmed cell death (PCD) has been well documented when Streptomyces species switch from vegetative (nutrition) to aerial (reproduction) growth, lethal determinants are yet to be discovered. Using DNA-damaging prodiginines of Streptomyces coelicolor as model system, we revealed that, under certain conditions, their biosynthesis is always triggered in the dying zone of the mycelial network prior to morphological differentiation, right after an initial round of cell death. The programmed massive death round of the vegetative mycelium is absent in a prodiginine non-producer (ΔredD strain), and mutant complementation restored both prodiginine production and cell death. The redD null mutant of S. coelicolor also showed increased DNA, RNA, and proteins synthesis when most of the mycelium of the wild-type strain was dead when prodiginines accumulated. Moreover, addition of the prodiginine synthesis inhibitors also resulted in enhanced accumulation of viable filaments. Overall, our data enable us to propose a model where the time-space production of prodiginines is programmed to be triggered by the perception of dead cells, and their biosynthesis further amplifies the PCD process. As prodiginine production coincides with the moment S. coelicolor undergoes morphogenesis, the production of these lethal compounds might be used to eradicate the obsolete part of the population in order to provide nutrients for development of the survivors. Hence, next to weapons in competition between organisms or signals in inter- and intra-species communications, we propose a third role for antibiotics (in the literal meaning of the word ‘against life’) i.e., elements involved in self-toxicity in order to control cell proliferation, and/or for PCD associated with developmental processes.

ANTIMICROBIAL PROPERTIES OF MYCELIUM AND BASIDIOCARPS OF GANODERMA LUCIDUM (СURTIS) P. KARST.

Aim. Evaluation of the reishi mushroom, Ganoderma lucidum, vegetative mycelium and water extracts of basidiocarps influence on the growth of opportunistic microorganisms. Materials and methods. Antimicrobial activity of vegetative mycelium and water extracts of basidiocarps from two strains of Ganoderma lucidum (Сurtis) P. Karst. ONU F101 and ONU F102 was screened by the agar diffusion method. Bacterial strains of Pseudomonas aeruginosa АТСС 27853, Proteus vulgaris ATCC 6896, Micrococcus luteus ATCC 4698, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212 and yeast-like fungus Candida albicans ATCC 18804 were used as the test-objects. Results. It was established that both reishi mushroom strains are able to inhibit the growth of opportunistic microorganisms under cultivation. Activity of water extracts was lower than vegetative mycelium of the fungus. The strains of different origin differ by the level and range of antimicrobial activity. Conclusions. The antimicrobial activity of the ONU F102 G. lucidum strain was lower than the ONU F101’s strain. The water extracts had less active activity compared with vegetative mycelium.

Streptomyces coelicolor in an oxygen-limited liquid environment: adapt and escape

Streptomycetes are mycelial soil bacteria that undergo a complex developmental cycle on solid media. Spores germinate and form a branched, vegetative mycelium. Several signals trigger the formation of hydrophobic aerial hyphae that differentiate further into reproductive chains of spores. Differentiation is accompanied by the production of secondary metabolites, e.g. antibiotics.

Sterigmatocystin production is restricted to hyphae located in the proximity of hülle cells.

Aspergillus nidulans produces sterigmatocystin, a secondary metabolite mycotoxin, for the protection of its reproductive structures. Previous studies on grazing behavior of fungivore arthropods, regulation of sexual development, and secondary metabolite biosynthesis have revealed the association of sterigmatocystin biosynthesis with sexual reproduction, but the spatial distribution of sterigmatocystin producing hyphae within the colony has never been investigated. In this work, we aimed to locate the site of sterigmatocystin production within the colony by employing a yCFP reporter system. We demonstrated that the stcO promoter is active only in vegetative hyphae that surround groups of hülle cells and the activity decreases and eventually ceases as the distance between the hypha and the hülle cells increases. This phenomenon indicates that the vegetative mycelium might consist of morphologically uniform, but functionally different hyphae.

Streptosporangium minutum sp. nov., isolated from garden soil exposed to microwave radiation.

The actinobacterium, strain M26T, was isolated from garden soil that was pre-treated with microwave radiation. The soil sample was collected in Roodepoort, Gauteng Province, South Africa as part of an antibiotic-screening programme. The isolate produced branched vegetative mycelium with sporangiophores bearing small sporangia ranging from 3 to 6 μm in diameter. Rapid genus identification revealed that the isolate belongs to the genus Streptosporangium. To confirm this result, the strain was subjected to polyphasic taxonomic characterisation. Chemotaxonomic characteristics were as follows: meso-DAP in the peptidoglycan, the whole-cell hydrolysate yielded madurose, predominant menaquinones were MK9 (21%), MK9(H2) (40%), MK9(H4) (31%) and MK9(H6) (3%); the polar lipid profile included an aminolipid, phosphoglycolipids, phosphatidylethanolamine, and phosphatidylmonomethylethanolamine. In addition, the fatty acid profile showed the presence of C16:0 (12.8%), C17:1ω8c (14.2%), and 10-methyl-C17:0 (15.8%). Furthermore, 16S rRNA gene sequence phylogenetic analysis showed that the strain is closely related to members of the genus Streptosporangium, which supports its classification within the family Streptosporangiaceae. Strain M26T exhibited antibiosis against a range of pathogenic bacteria, including, but not limited to Acinetobacter baumannii ATCC 19606T, Enterobacter cloacae subsp. cloacae ATCC BAA-1143, Enterococcus faecalis ATCC 51299 (vancomycin resistant), Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19111, Mycobacterium tuberculosis H37RvT, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica subsp. arizonae ATCC 13314T, and the methicillin-resistant Staphylococcus aureus subsp. aureus ATCC 33591 (MRSA). The name Streptosporangium minutum is proposed with the type strain M26T (=LMG 28850T =NRRL B-65295T).

Non-targeted Colonization by the Endomycorrhizal Fungus, Serendipita vermifera, in Three Weeds Typically Co-occurring with Switchgrass.

Serendipita vermifera (=Sebacina vermifera; isolate MAFF305830) is a mycorrhizal fungus originally isolated from the roots of an Australian orchid that we have previously shown to be beneficial in enhancing biomass yield and drought tolerance in switchgrass, an important bioenergy crop for cellulosic ethanol production in the United States. However, almost nothing is known about how this root-associated fungus proliferates and grows through the soil matrix. Such information is critical to evaluate the possibility of non-target effects, such as unintended spread to weedy plants growing near a colonized switchgrass plant in a field environment. A microcosm experiment was conducted to study movement of vegetative mycelia of S. vermifera between intentionally inoculated switchgrass (Panicum virgatum L.) and nearby weeds. We constructed size-exclusion microcosms to test three different common weeds, large crabgrass (Digitaria sanguinalis L.), Texas panicum (Panicum texanum L.), and Broadleaf signalgrass (Brachiaria platyphylla L.), all species that typically co-occur in Southern Oklahoma and potentially compete with switchgrass. We report that such colonization of non-target plants by S. vermifera can indeed occur, seemingly via co-mingled root systems. As a consequence of colonization, significant enhancement of growth was noted in signalgrass, while a mild increase (albeit not significant) was evident in crabgrass. Migration of the fungus seems unlikely in root-free bulk soil, as we failed to see transmission when the roots were kept separate. This research is the first documentation of non-targeted colonization of this unique root symbiotic fungus and highlights the need for such assessments prior to deployment of biological organisms in the field.

14-3-3 proteins are involved in growth, hyphal branching, ganoderic acid biosynthesis, and response to abiotic stress in Ganoderma lucidum.

Ganoderma lucidum, which contains many pharmacologically active compounds, is regarded as a traditional medicinal fungus. Nevertheless, the scarcity of basic research limits the commercial value and utilization of G. lucidum. As a class of highly conserved, phosphopeptide-binding proteins present in all eukaryotes, 14-3-3 proteins play vital roles in controlling multiple physiological processes, including signal transduction, primary metabolism, and stress responses. However, knowledge of the roles of 14-3-3 proteins in Basidiomycetes is sparse. In this article, two homologs of 14-3-3 proteins, encoded by the two distinct genes GlBmh1 and GlBmh2, were distinguished in G. lucidum. We found that GlBmh1 and GlBmh2 were expressed at various developmental stages, including in vegetative mycelium cultivated on solid medium and in primordia and fruiting bodies. Moreover, we constructed GlBmh1 single-silenced strains, GlBmh2 single-silenced strains, and 14-3-3 double-silenced mutants for further study. When GlBmh1 and GlBmh2 were inhibited by RNA interference, the growth rate of mycelia was decreased, and the distance between the aerial hyphal branches was reduced; responses to various abiotic stresses such as oxidants and cell wall and osmotic stressors were also changed. Furthermore, the contents of secondary metabolite ganoderic acids (GAs) were increased after GlBmh1 and GlBmh2 were simultaneously silenced. Taken together, we provide evidence that implicates potential roles for the two 14-3-3 proteins in affecting growth and GA biosynthesis, thereby providing new insights into the basic functions of 14-3-3 proteins in G. lucidum.

Bioaccumulation of Lithium (Li2CO3) in Mycelia of the Culinary-Medicinal Oyster Mushroom, Pleurotus ostreatus (Agaricomycetes).

Pleurotus ostreatus is a white-rot mushroom that bioaccumulates metals in basidiocarps and vegetative mycelia. This fungus has been used in soil and water bioremediation of several heavy metals; however, there are few studies of lithium mycelial bioaccumulation for pharmacological use. The aim of this study was to evaluate lithium bioaccumulation in P. ostreatus mycelia grown in a liquid malt extract cultivation medium with Li2CO3 or LiCl. Each lithium source was added to the medium to obtain a concentration of 0, 5, 10, 15, 20, 25, 30, 40, 50, 100, or 200 mg · L-1 lithium. The highest bioaccumulation of lithium in mycelia was 1575.29 μg · g-1 upon treatment with 40 mg · L-1 Li2CO3. P. ostreatus mycelia produce biomass and bioaccumulate both lithium sources, but more lithium bioaccumulates when in the form of Li2CO3. This study provides a prospective for the development of biotechnological products with high aggregate values and alternative ways to deliver lithium and eventually other salts for pharmacological use.

Морфолого-культуральні властивості рідкісного лікарського гриба Sparassis crispa (Sparassidaceae, Polyporales)

<p>A verification of <em>S</em><em>parassis</em><em> crispa</em><em> </em>(Wulfen) Fr. strains of the IBK Mushroom Culture Collection using molecular-genetic and cultural-morphological methods were done. For all <em>S. crispa</em> strains a complete determination of the nucleotide sequences of the internal transcribed spacer: ITS1, 5.8S and ITS2 regions of rRNA, as well as the partial determination of 18S and 28S sequences surrounding the ITS, was performed. As a result of the search in the gene bank, the <em>S</em><em>. </em><em>crispa</em> samples deposited there showed 99-98% identity with the sequences we received, thus confirming the species of the examined strains. On the basis of the sequences we received, investigated strains were registered in NCBI GenBank. To confirm the taxonomic affiliation of the strains we studied their cultural and morphological characteristics. Microstructures of vegetative mycelium by optical and scanning electron microscopy were investigated. In all <em>S. crispa</em> cultures we observed the hyphae with regular one-sided gapless clamp connections, numerous secretory cells on the surface of the hyphae, anastomoses, filamentous strands and films. We studied the growth rate and morphology of the strains on seven agar nutrient media. According to the radial growth rate of <em>S. crispa</em> cultures can be placed to the very slowly growing mushrooms, growth rate of 0,5−2,8 mm/day. We found the selective media such as malt agar with the addition of pine sawdust (SS) and larch sawdust (SM) as most favorable for the vegetative growth and generative stage for all strains, temperature of incubation was 26 ± 0.1 °C. For mycelial growth the critical temperature was 39 ± 0.1 °C for <em>S. crispa</em> strains 312, 314 and 40 ± 0.1 °C for the strains 304, 2004. We confirmed belonging of studied strains to <em>S. </em><em>с</em><em>rispa</em> species according to established morphological and cultural characteristics. This result coincided with the results of DNA typing. Thus, the obtained mycelium growth parameters on nutrient media, micro- and macromorphological characteristics can be used as additional taxonomic characteristics of <em>S. </em><em>с</em><em>rispa</em> culture in the vegetative stage of growth.<strong> </strong>The strain <em>S. </em><em>с</em><em>rispa</em> 314 may become a potential producer for new fungal biotechnologies in Ukraine in the near future and reintroduction of this species in the nature.</p>

Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria

Armillaria species are both devastating forest pathogens and some of the largest terrestrial organisms on Earth. They forage for hosts and achieve immense colony sizes via rhizomorphs, root-like multicellular structures of clonal dispersal. Here, we sequenced and analysed the genomes of four Armillaria species and performed RNA sequencing and quantitative proteomic analysis on the invasive and reproductive developmental stages of A. ostoyae. Comparison with 22 related fungi revealed a significant genome expansion in Armillaria, affecting several pathogenicity-related genes, lignocellulose-degrading enzymes and lineage-specific genes expressed during rhizomorph development. Rhizomorphs express an evolutionarily young transcriptome that shares features with the transcriptomes of both fruiting bodies and vegetative mycelia. Several genes show concomitant upregulation in rhizomorphs and fruiting bodies and share cis-regulatory signatures in their promoters, providing genetic and regulatory insights into complex multicellularity in fungi. Our results suggest that the evolution of the unique dispersal and pathogenicity mechanisms of Armillaria might have drawn upon ancestral genetic toolkits for wood-decay, morphogenesis and complex multicellularity.

Nuclear populations of the multinucleate fungus of leafcutter ants can be dekaryotized and recombined to manipulate growth of nutritive hyphal nodules harvested by the ants

ABSTRACTWe dekaryotized the multinucleate fungus Leucocoprinus gongylophorus, a symbiotic fungus cultivated vegetatively by leafcutter ants as their food. To track genetic changes resulting from dekaryotization (elimination of some nuclei from the multinuclear population), we developed two multiplex microsatellite fingerprinting panels (15 loci total), then characterized the allele profiles of 129 accessions generated by dekaryotization treatment. Genotype profiles of the 129 accessions confirmed allele loss expected by dekaryotization of the multinucleate fungus. We found no evidence for haploid and single-nucleus strains among the 129 accessions. Microscopy of fluorescently stained dekaryotized accessions revealed great variation in nuclei number between cells of the same vegetative mycelium, with cells containing typically between 3 and 15 nuclei/cell (average = 9.4 nuclei/cell; mode = 8). We distinguish four mycelial morphotypes among the dekaryotized accessions; some of these morphotypes had lost the full competence to produce gongylidia (nutritive hyphal-tip swellings consumed by leafcutter ants as food). In mycelial growth confrontations between different gongylidia-incompetent accessions, allele profiles suggest exchange of nuclei between dekaryotized accessions, restoring full gongylidia competence in some of these strains. The restoration of gongylidia competence after genetic exchange between dekaryotized strains suggests the hypothesis that complementary nuclei interact, or nuclear and cytoplasmic factors interact, to promote or enable gongylidia competence.

Lectins from Mycelia of Basidiomycetes.

Lectins are proteins of a nonimmunoglobulin nature that are capable of specific recognition of and reversible binding to the carbohydrate moieties of complex carbohydrates, without altering the covalent structure of any of the recognized glycosyl ligands. They have a broad range of biological activities important for the functioning of the cell and the whole organism and, owing to the high specificity of reversible binding to carbohydrates, are valuable tools used widely in biology and medicine. Lectins can be produced by many living organisms, including basidiomycetes. Whereas lectins from the fruit bodies of basidiomycetes have been studied sufficiently well, mycelial lectins remain relatively unexplored. Here, we review and comparatively analyze what is currently known about lectins isolated from the vegetative mycelium of macrobasidiomycetes, including their localization, properties, and carbohydrate specificities. Particular attention is given to the physiological role of mycelial lectins in fungal growth and development.

The roles of actin cytoskeleton and actin-associated protein Crn1p in trap formation of Arthrobotrys oligospora

Nematode-trapping fungi include a variety of species capable of generating specific trapping devices to capture nematodes and the production of devices is also an indicator of a switch from saprophytic to predacious lifestyles. Traps are developed from vegetative mycelia, but they are quite different from hyphae in both morphological and physiological characteristics. Therefore, the molecular mechanisms underlying their formation have attracted much attention. In this investigation, Arthrobotrys oligospora, a nematode-trapping fungus, has three-dimensional networks and genomics and proteomics were recently performed, so as to reveal the relationship between actin cytoskeleton and trap formation. Both actin staining via FITC-phalloidin and treatment of actin polymerization inhibitor Lat-B illustrated that the actin cytoskeleton played an important role in trap development. Furthermore, absence of the conserved actin-associated protein Crn1p caused a structural defect of traps and failure to infect nematodes. It was observed that mutant Δcrn1 represented a reduced number of rings and a lower complexity of three-dimensional networks, likely due to the disturbance of actin branching. Collectively, our study confirmed the involvement of the actin cytoskeleton as well as the conserved actin-associated protein Crn1p in trap formation. It further suggested the manners in which Crn1p influences the development of three-dimensional networks in A. oligospora.

The 25-26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes.

Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host–pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25–26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25–26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25–26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25–26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora.

Evaluation of casing variables for cultivation of Agaricusbisporus strain U3 in Punjab, India

Casing is an important step during cultivation of button mushroom. It promotes a shift of vegetative mycelium to form pinheads leading to mature basidiocarps. Therefore, An experiment was planned at Punjab Agricultural University, Ludhiana in which farm yard manure (FYM), biogas slurry (BS), burnt rice husk (BRH), spent compost (SC), coir pith (CP) and sandy soil (SS) were used in six combinations to evaluate their impact on yield of Agaricusbisporus strain U3. Casing mixtures were analyzed for the moisture, water holding capacity (WHC), bulk density, pH, electrical conductivity (EC) and mineral analysis. The moisture content of casing mixtures ranged between 33 to 56.3% with maximum in FYM+CP (1:1) and FYM+BS (1:1) which results in more water holding capacity with 160% and 100% for FYM+CP (1:1) and FYM+BS (1:1) respectively. The bulk density of different casing mixtures ranged between 33.3 to 83.3% and maximum for FYM+BS as well as the control FYM+SS (4:1). No significant variation in pH of casing mixture was observed while EC was significantly higher in FYM+BS (1:1) with 1.7 mmhos cm-1. Microbial count of different casing mixtures was between 3.0-5.7×105 which was statistically non-significant with respect to casing mixtures. A mixture of FYM+BRH (1:1) gave maximum yield of 15.1 kg with heavy fruit bodies. Number of fruiting bodies in casing mixture FYM+BRH (1:1) were 31.4, 26.4, 148.1,31.4 and 8.1 percent higher over FYM+CP (1:1), FYM+SC (1:1), FYM+BS (1:1), FYM and FYM+SS (4:1,control), respectively. The casing mixture FYM+BRH (1:1) with higher number of fruiting bodies with maximum fruiting bodies weight was the best alternate in mushroom growing industry.

English

The role of the ectomycorrhizal (Myc) fungus Suillus luteus as a biological control agent against damping off caused by Fusarium verticillioides (Fo) and Fusarium oxysporum (Fm) on Scots (Pinus sylvestris L.) and Stone pine (Pinus pinea L.) was studied in a greenhouse experiment. The vegetative mycelium of S. luteus in a vermiculite/peat carrier was added to potting substrate before inoculation with Fo or Fm spores (macro and microconidia). Also, seedlings were inoculated only with the Myc, Fo, Fm or water for treatment comparisons. The seedling disease index (SDI) of seedlings varied significantly among pine, Fusarium and Myc treatments. Scots pine seedlings inoculated with F. verticillioides and F. oxysporum had a reduced SDI when co-cultured with S. luteus. Damage in Stone pine seedlings inoculated with F. oxysporum was significantly reduced in the presence of Myc fungus, but no reduction of disease symptoms was observed when inoculated with F. verticillioides. Mycorrhizal formation in co-cultures with F. verticillioides was low and absent in co-cultures with F. oxysporum, although S. luteus inoculation resulted in a greater antagonism against this latter pathogen. The protective effect of S. luteus against damping-off by Fusarium species was not related to the percent of mycorrhizal apexes in the roots of Stone and Scots pine seedlings. Key words: Forest nurseries, ectomycorrhiza, Suillus luteus, damping off, Fusarium oxysporum, Fusarium verticillioides, biological control, Pinus species.

(1→3)-α-d-Glucan from Fruiting Body and Mycelium ofCerrena unicolor(Bull.) Murrill: Structural Characterization and Use as a Novel Inducer of Mutanase

Water-insoluble, alkali-soluble polysaccharide (marked as ASP) was extracted from the vegetative mycelium and fruiting body ofCerrena unicolorstrain. Monosaccharide examination of ASP demonstrated that the isolated biopolymer was composed mainly of glucose, xylose, and mannose monomers. The methylation investigation of studied polymers indicated that (1→3)-linkedα-D-Glcpis the major chain constituent (92.2% for glucans isolated from fruiting body and 90.1% from mycelium).1H NMR, FT-IR, and immunofluorescent labelling determinations confirmed that the polysaccharides isolated from both fruiting body and mycelium ofC. unicolorare (1→3)-α-d-glucans. The obtained (1→3)-α-d-glucans showed differences in viscosity and similar characteristics in optical rotations. (1→3)-α-d-Glucans extracted from mycelium and fruiting body ofC. unicolorwere also used as potential and specific inducers of mutanase synthesis byTrichoderma harzianum. The highest mutanase activity (0.38 U/mL) was obtained after induction of enzyme by (1→3)-α-d-glucan isolated from the mycelium ofC. unicolor, and this biopolymer has been suggested as a new alternative to streptococcal mutan for the mutanase induction inT. harzianum. (1→3)-α-d-Glucan-induced mutanase showed high hydrolysis potential in reaction with dextranase-pretreated mutan, where maximal degree of saccharification and solubilization of this bacterial homoglucan (83.1% and 78.4%, resp.) was reached in 3 h at 45°C.

Characterization and phylogenetic affiliation of Actinobacteria from tropical soils with potential uses for agro-industrial processes.

Secondary metabolites produced by Actinobacteria of tropical soils represent a largely understudied source of novel molecules with relevant application in medicine, pharmaceutical and food industries, agriculture, and environmental bioremediation. The present study aimed to characterize sixty-nine Actinobacteria isolated from compost and tropical soils using morphological, biochemical, and molecular methods. All the isolates showed high variation for morphological traits considering the color of pigments of the aerial and vegetative mycelium and spore chain morphology. The enzymatic activity of amylase, cellulase, and lipase was highly variable. The amylase activity was detected in 53 (76.81%) isolates. Eighteen isolates showed enzymatic index (EI) > 4.0, and the isolates ACJ 45 (Streptomyces curacoi) and ACSL 6 (S. hygroscopicus) showed the highest EI values (6.44 and 6.42, respectively). The cellulase activity varied significantly (P ≤ 0.05) among the isolates. Twenty-nine isolates (42.02%) showed high cellulase activity, and the isolates ACJ 48 (S. chiangmaiensis) and ACJ 53 (S. cyslabdanicus) showed the highest EI values (6.56 for both isolates). The lipase activity varied statistically (P ≤ 0.05) with fourteen isolates (20.29%) considered good lipase producers (EI > 2.0). The isolate ACSL 6 (S. hygroscopicus) showed the highest EI value of 2.60. Molecular analysis of partial 16S rRNA gene sequencing revealed the existence of 49 species, being 38 species with only one representative member and 11 species represented by one or more strains. All species belonged to three genera, namely Streptomyces (82.61%), Amycolatopsis (7.25%), and Kitasatospora (10.14%). The present results showed the high biotechnological potential of different Actinobacteria from tropical soils.