A reappraisal of the Pleurotus eryngii complex – New species and taxonomic combinations based on the application of a polyphasic approach,and an identification key to Pleurotus taxa associated with Apiaceae plants

The Pleurotus eryngii species-complex comprises choice edible mushrooms growing on roots and lower stem residues of Apiaceae (umbellifers) plants. Material deriving from extensive sampling was studied by mating compatibility, morphological and ecological criteria, and through analysis of ITS1-5.8S-ITS2 and IGS1 rRNA sequences. Results revealed that P. eryngii sensu stricto forms a diverse and widely distributed aggregate composed of varieties elaeoselini, eryngii, ferulae, thapsiae, and tingitanus. Pleurotuseryngii subsp. tuoliensis comb. nov. is a phylogenetically sister group to the former growing only on various Ferula species in Asia. The existence of Pleurotusnebrodensis outside of Sicily (i.e., in Greece) is reported for the first time on the basis of molecular data, while P. nebrodensis subsp. fossulatus comb. nov. is a related Asiatic taxon associated with the same plant (Prangos ferulacea). Last, Pleurotusferulaginis sp. nov. grows on Ferulago campestris in northeast Italy, Slovenia and Hungary; it occupies a distinct phylogenetic position accompanied with significant differences in spore size and mating incompatibility versus other Pleurotus populations. Coevolution with umbellifers and host/substrate specificity seem to play key roles in speciation processes within this fungal group. An identification key to the nine Pleurotus taxa growing in association with Apiaceae plants is provided.     

Genetic polymorphism of ferula mushroom growing on Ferula sinkiangensis

Mating tests, internal transcribed spacer (ITS) sequence analysis, intergenic spacer 1–restriction fragment length polymorphism (IGS1-RFLP), IGS1 sequence analysis, and IGS2-RFLP analysis were carried out on isolates of 17 morphologically different Pleurotus mushrooms collected on Ferula sinkiangensis. The isolates were divided, based on mating tests and ITS sequence analysis, into two groups identical to P. eryngii var. ferulae and P. nebrodensis, respectively. Single spores from these two groups were incompatible, but those from P. eryngii var. ferulae and P. eryngii were compatible and combined to produce 56.25% dikaryon mycelia with clamp connections. The ITS of P. eryngii var. ferulae and P. nebrodensis (GenBank accession no. AY311408) were both 638 bp in size but differed by 3% in sequence. P. eryngii var. ferulae and P. eryngii (GenBank accession no. AY368658) were identical in ITS size and sequence. P. nebrodensis was the dominant population of Pleurotus mushroom growing on F. sinkiangensis. It exhibited genetic diversity. The two species could also be distinguished by IGSI-RFLP, similar to identification by mating tests and ITS sequence analysis. Difference in IGS1-RFLP existed between P. eryngii var. ferulae and P. nebrodensis. The sequence difference reached 2.28%. Both IGS1 size and IGS1-RFLP were similar among the different samples of P. nebrodensis. The 17 isolates were separated into five types based on IGS2 size and IGS2-RFLP, with both interspecies and extraspecies differences. P. nebrodensis exhibited polymorphism and was divided into four types. These results agreed with macroscopic differences. IGS2 might be the effective domain of genetically polymorphic ribosomal DNA in P. nebrodensis mushrooms found in Xinjiang, China.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Taxonomic position of a Chinese <Emphasis Type="Italic">Pleurotus</Emphasis> “Bai-Ling-Gu”: it belongs to <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> (DC.: Fr.) Quél. and evolved independently in China

The Chinese Bai-Ling-Gu is a mushroom named Pleurotus eryngii var. tuoliensis C.J. Mou. This species has been identified as P. nebrodensis or P. eryngii var. nebrodensis. We examined its taxonomic position by analysis of mating, cultivation, and rDNA sequences, and concluded as follows. (1) Bai-Ling-Gu mated with P. eryngii var. eryngii, and the F₁ and F₂ formed fruit bodies. (2) Bai-Ling-Gu mated with P. eryngii var. ferulae, and the F₁ formed fruit bodies. (3) In the di-mon mating test, P. eryngii var. nebrodensis from Sicily mated with monokaryons of P. eryngii var. eryngii but mated hardly at all with those of Bai-Ling-Gu and P. eryngii var. ferulae. The di-mon mating pattern of Bai-Ling-Gu resembled those of P. eryngii var. ferulae. (4) The partial sequences of rDNA ITS1 and IGS1 from the epitype of P. nebrodensis were identical with those from P. eryngii var. nebrodensis from Sicily but differed from those from Bai-Ling-Gu. (5) The strains of P. eryngii var. eryngii and P. eryngii var. ferulae were in a group, the strains of P. eryngii var. nebrodensis from Sicily were in another group, and the strains of Bai-Ling-Gu were in the other group in both the phylogenetic trees based on the ITS1 and the IGS1 sequences. These results led to the conclusion that Bai-Ling-Gu is a variety of P. eryngii and evolved independently in China. It is satisfactory to identify Bai-Ling-Gu with P. eryngii var. tuoliensis C.J. Mou.     

Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita,Volvariella volvacea and Pleurotus spp.

Ten selected wild and commercial strains of Pleurotus ostreatus,Pleurotus eryngii,Pleurotus pulmonarius, Agrocybe aegerita andVolvariella volvacea were cultivated on three agricultural wastes, i.e. wheat straw (WS), cotton waste (CW) and peanut shells (PS). All species demonstrated significantly higher colonization rates on WS and CW than on PS. WS supported faster growth of A. aegerita and Pleurotus spp., whereas V. volvacea performed better on CW. Comparison of growth rates on composted and non-composted WS and CW substrates revealed that in the latter case faster colonization was achieved, particularly for Pleurotus spp. However, one commercial strain of V. volvacea presented higher growth rates when the composted CW medium was used. Furthermore, earliness in the fructification of P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW substrates, while WS favoured earliness of P. eryngii and A. aegerita. Similarly, high sporophore yields were obtained by P. ostreatus and P. pulmonarius on both wastes, whereas WS enhanced yield and basidioma size of P. eryngii and A. aegerita strains and CW production of V. volvacea. The substrates cellulose:lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of P. ostreatus and P. pulmonarius; in addition, positive correlation was also detected for carbon:nitrogen ratio and mushroom yield in P. eryngii and A. aegerita and between cellulose content and mushroom yield for V. volvacea strains.     

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> complex

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man’s selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods—amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes—to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.     

Edible mushrooms from olive oil mill wastes

The biological remediation of olive oil mill wastes has been attempted several times in the past through the use of different types of microbes. Among them, a relatively large array of fungi were studied for neutralizing the heavy pollutant effects and/or for converting these wastes into new value-added products. The present investigation was aiming at examining whether olive oil mill wastes could be exploited for the cultivation of mushrooms of the genus Pleurotus. At a preliminary stage, two Pleurotus species, i.e. P. eryngii and P. pulmonarius, were tested for their ability to colonize an olive press-cake (OPC) substrate supplemented with various dilutions of raw olive mill wastewater (OWW). Some important cultural characters related to mushroom production (earliness, yield, biological efficiencies and quality of basidiomata) were estimated. The outcome revealed different cultural responses for each Pleurotus species examined; the P. pulmonarius strain showed better earliness values and P. eryngii, although it was a slow growing fungus, produced basidiomata in high yields and of a very good quality. On the other hand, the OPC substrate supplemented with low concentrations of OWW (12.5% v/w) behaved satisfactorily as regards the fungal colonization rates and mushroom yield, but when the addition of higher rates of raw, untreated OWW (75–100% v/w) was attempted then the Pleurotus strains were completely unable to grow. The optimal concentration of OWW for Pleurotus mycelial growth was assessed through measurements of the biomass produced in liquid nutrient media and was found to lie within the 25–50% range, depending on the Pleurotus species and on the properties of the substrates examined. Furthermore, the phytotoxic effects that the spent liquid medium possessed were examined in comparison with the phytotoxicity of the raw liquid waste. The prospects of exploiting olive oil mills wastes for mushroom cultivation is discussed.     

Molecular characterization and mating type analysis of oyster mushroom (<Emphasis Type="Italic">Pleurotus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>) using single basidiospores for strain improvement

Existence of variability in morphological traits and growth rate of mycelium of homokaryotic single basidiospores can be exploited for the development of inter-strainal hybrids. We isolated 182 single basidiospores from mushroom bodies of P. sajor-caju, P. florida, P. eous and one wild relative of Pleurotus called Hypsizygus ulmaris. The single spores were isolated using a new technique that is less prone to contamination and more efficient than the common techniques used by earlier workers. All the isolates showed a varied range of cultural morphology. Mating types of all the isolates within the species were identified on the basis of hyphal fusion via anastomosis with the tester strains. Four compatible pairs of isolates with well prominent tuft in the contact zone were selected for dikaryon isolation. Dikaryons were used for spawn preparation and mushroom cultivation. The dikaryotic isolates with their replicates were evaluated for spawn run period, yield and biological efficiency. 42 isolates (10 di- and 32 mono-karyotic isolates) were analyzed with RAPD genetic markers. Phenotypic characters and mating types of all the 42 isolates analyzed genetically were correlated with their genetic polymorphism data. The isolates showed very large diversity both at the phenotypic and the genotypic level. Available phenotypic and genotypic data can further help in the selection of monosporous isolates for developing inter-strainal hybrids which can lead to better prospects for genetic improvement in different species of Pleurotus.     

Gene cloning,heterologous expression,in vitro reconstitution and catalytic properties of a versatile peroxidase

The gene of a peroxidase described as being involved in carotenoid degradation was cloned from a strain that was conserved as Lepista irina (CBS 458.79). Gene sequencing revealed high nucleotide and amino-acid identity with Pleurotus eryngii gene vpl, which encodes a versatile peroxidase with unique catalytic properties, and only reported in Pleurotus and Bjerkandera species. Re-identification of the supposed L. irina strain revealed that, in fact, it is a P. eryngii strain. The new P. eryngii peroxidase was expressed in Escherichia coli, and the recombinant protein folded in the presence of cofactor to obtain the active form. The purified enzyme was able to oxidize Mn²⁺, veratryl alcohol, substituted phenols, and both low and high redox-potential dyes, demonstrating that it belongs to the versatile peroxidase family (named VPL3). These catalytic properties agreed with the presence of both Mn²⁺ and aromatic-substrate oxidation sites in its molecular structure.     

A new species of Pithoascus and first report of this genus as endophyte associated with Ferula ovina

A newly described species of Pithoascus from roots of Ferula ovina differs from other Pithoascus species by producing larger ascomata than all described species except P. exsertus. The shape of its ascospores is similar to that of P. lunatus, but differs from it by having an asexual state. This new species differs from P. ater by having a sexual state. Phylogenetic analyses based on concatenated ITS rDNA, LSU rDNA and partial translation elongation factor 1 alpha gene datasets also confirmed the generic placement in Pithoascus and showed its close phylogenetic relationships to P. ater and P. lunatus.     

Mitochondrial morphogenesis and ultrastructure of basidiomycetes from genera Agaricus and Pleurotus

Supravital study of rhodamine-stained mitochondria in cells of aerial and submerged micelium of 31 strains from 9 species from genus Agaricus (A. arvensis Schaeff., A. bisporus (Lange) Imbach, A. bitorquis (Quel.) Sacc., A. campestris L., A. excellens, (F.H. Müller) F.H. Müller, A. macrocarpus (F.H. Müller) F.H. Müller, A. silvaticus Schaeff., A. silvicola (Vittad.) Peck, A. xanthodermus Genev) and of 2 strains from 2 species from genus Pleurotus (P. ostreatus (Jacg.) P. Kumm., P. pulmonarius (Fr.) Quel.) was carried out. Mitochondrial morphogenesis in micelial cells of species from Agaricus and Pleurotus genera has many common features in distribution of mitochondria in micelial cells both under the favorable growth conditions and during chondriom reorganization (fission and fragmentation of mitochondria to small subunits) under unfarovable growth conditions and during aging. During the balanced growth of mycelium of heterokaryotic strains from genera Agaricus and Pleurotus for 7–14 days on agar media as well as during cultivation of mycelium of oyster mushroom and several strains of field mushroom in submerged culture, distribution of mitochondria by the type 1 (small granular mitochondria in the apical zone—1, long rod-like mitochondria in the subapical zone—2 and short rod-like and granular mitochondria formed as a result of fragmentation of rod-like mitochondria in mature mycelium cells—3) was observed. Under unfarovable growth conditions (starvation), in mycelium cells of homokaryotic strains of champignon, during long cultivation of all strains and species from the studied genera and during cultivation on liquid wort, for majority of champignon, distribution of mitochondria by the type 2 was observed (sphere-like mitochondria in all cells of the studied mycelium zones). Mitochondrial profiles at ultrastructural level had specific features, such as associations of the outer mitochondrial membrane with cytoplasmic ribosomes and changes in cristae structure. The preliminary test for apoptosis-like phenotype of the submerged mycelium cells of Bs94 champignon that hardly grows in the submerged culture gave positive result. Thus, mitochondrial morphogenesis in mycelium cells of Agaricus and Pleurotus species under different conditions and terms of cultivation occurs similarly and is determined by a complex of physiological and biochemical processes reflecting the state of mycelium cells.     

Atypical non-lipid-dependent strains of <Emphasis Type="Italic">Malassezia furfur</Emphasis>

Members of the yeast genus Malassezia, including atypical strains, are lipophilic except for Malassezia pachydermatis. New physiological features that characterize atypical Malassezia strains are mainly associated with alteration in Tween assimilation pattern — such isolates still require lipids for growth. We isolated three non-lipid-dependent strains of Malassezia from patients with diagnosed atopic dermatitis (AD). These isolates could not be identified to the species level via their physiological properties. Phylogenetic trees, based on the D1/D2 regions of the 26S rDNA gene sequences and nucleotide sequences of the ITS1-5.8S-ITS2 rRNA region, showed the isolates to belong to Malassezia furfur. Three non-lipid dependent isolates from AD skin were conspecific, and sequences analysis proved them to be M. furfur.     

Cultivation of different strains of king oyster mushroom (Pleurotus eryngii) on saw dust and rice straw in Bangladesh

Pleurotus eryngii is a popular mushroom due to its excellent consistency of cap and stem, culinary qualities and longer shelf life. In Bangladesh, where Pleurotus mushrooms are very popular, P. eryngii may take position among the consumers, but currently this mushroom is not cultivated in large scale there. In this study, 3 strains of P. eryngii such as Pe-1 (native to Bangladesh), Pe-2 (germplasm collected from China) and Pe-3 (germplasm collected from Japan) were cultivated on saw dust and rice straw and their growth and yield parameters were investigated. Pe-1 on saw dust showed the highest biological yield and efficiency (73.5%) than other strains. Also, the mycelium run rate and number of fruiting bodies were higher in Pe-1 than other two strains. The quality of mushroom strains was near about similar. On saw dust, the yield and efficiency were better than those cultivated on rice straw, however, on straw; the mushroom fruiting bodies were larger in size. This study shows the prospects of P. eryngii cultivation in Bangladesh and suggests further study in controlled environment for higher yield and production.     

Effect of antimicrobial activity of Melaleuca alternifolia essential oil on antagonistic potential of Pleurotus species against Trichoderma harzianum in dual culture

Melaleuca alternifolia (tea tree) essential oil was investigated for its “in vitro” ability to control Trichoderma harzianum, a fungal contaminant that causes extensive losses in the cultivation of Pleurotus species. The antifungal activity of M. alternifolia essential oil and antagonist activities between Pleurotus species against three T. harzianum strains were studied in dual-culture experiments on an agar-based medium in which different concentrations of essential oil were incorporated. M. alternifolia essential oil at a concentration of 0.625 μL/mL, inhibited T. harzianum mycelial growth by 5.9–9.0%, depending on the strain. At the same concentrations P. ferulae and P. nebrodensis stimulated mycelial growth by 5.2–8.1%. All strains of T. harzianum were antagonistic to the Pleurotus species in the control. When essential oil was added to the substrate cultural, the antagonistic activity of T. harzianum against the Pleurotus species was weak (0.0625 μL of essential oil) or non-existent (0.125 μL of essential oil). M. alternifolia essential oil could be an alternative to the synthetic chemicals that are currently used to prevent and control T. harzianum in mushroom cultivation.     

Phylogenetic positions of seven poorly known species of Ferula (Apiaceae) with remarks on the phylogenetic utility of the plastid trnH-psbA,trnS-trnG,and atpB-rbcL intergenic spacers

Ferula L. is one of the most species-rich and taxonomically difficult genera of Apiaceae. In this study, we obtained nrDNA ITS sequences of seven poorly known species of Ferula (Ferula anatolica, Ferula sp. (tentatively identified as F. candelabrum by collectors), F. drudeana, F. huber-morathii, F. marmarica, F. talassica, and F. tunetana) and explored their phylogenetic positions using 148 ITS sequences of the subtribe Ferulinae from GenBank. Five of these newly sequenced species fall into three groups, corresponding to clades recognized in earlier molecular studies. Ferula sp. are added to clade, which is mostly composed of Central Asian species. This placement showed that identification as F. candelabrum was erroneous. The second clade, which is mostly composed of Mediterranean taxa, includes two species from North Africa: F. marmarica and F. tunetana. Despite the well-supported monophyly of this clade, the relationships inside this group need to be revised, as broadly distributed F. communis is paraphyletic with respect to other species. Ferula drudeana and F. huber-morathii, two narrow endemics from Turkey, are placed in the Central Asian clade. Two species, F. anatolica and F. talassica, do not fall into any of the recognized clades. In addition, we examined the sequence variation of three potentially highly variable pDNA regions, the trnH-psbA, trnS-trnG, and atpB-rbcL intergenic spacers, for a subset of 18 specimens. The resulting pDNA and ITS based phylogenetic trees were incongruent, as supported by significant ILD tests. The cause of this incongruence can be manifold, including hybridization, a lack of a phylogenetic signal, and homoplastic substitutions. Our analyses suggest that only trnS-trnG can be added to the list of pDNA markers used for phylogenetic studies of Ferula, as it has the highest number of parsimony informative characters and is easy to amplify from degraded material.     

Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5.8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.     

Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: Structure and potential prebiotic activity

Cultivated oyster mushrooms (genus Pleurotus) are interesting as a source of biologically active glucans. Partially, β-glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. Specific glucans were isolated from stems of Pleurotus ostreatus and Pleurotus eryngii by subsequent boiling water and alkali extraction. Obtained water soluble (L1), alkali soluble (L2) and insoluble (S) fractions were characterised by various analytical methods. Spectroscopic analysis detected glucans in all the fractions: branched 1,3-1,6-β-d-glucan predominated in L1 and S, while linear 1,3-α-d-glucan in L2. Fractions L1 also contained marked amount of proteins partially in complex with glucans; protein content in L2 was insignificant. Effective deproteinisation of L1 and separation of α- and β-glucans in L2 was achieved by the treatment with phenolic reagent. Small amount of chitin was found in S as a component of cell wall chitin–glucan complex. Potential prebiotic activity of extracts L1 and L2 was testing using nine probiotic strains of Lactobacillus, Bifidobacterium and Enterococcus. These probiotics showed different growth characteristics dependently on used extract and strain specificity due to the presence of structurally diverse compounds. The extracts L1 and L2 can be applied to synbiotic construction only for carefully selected probiotic strains. This exploitation of fruit body extracts extends the use of mushrooms P. ostreatus and P. eryngii for human health.     

Decolorization of olive oil mill liquid wastes by chemical and biological means

The removal of colour from olive mill wastewaters (OMW) using aluminium sulphate [Al₂(SO₄)₃·18H₂O], lime (CaO) and hydrogen peroxide (H₂O₂) was investigated, along with the decolorizing effect of sixteen strains of Pleurotus. In general terms all chemical substances exerted a clear decolorizing effect, yet the most effective was hydrogen peroxide, followed by lime and alumina. The tests with Pleurotus isolates were carried out on plates using different concentrations (25%, 50%, 75%, 100%) of sterilized OMW solidified with 1.5% agar. For all strains tested, decolorization proceeded more slowly than radial growth. Among the six Pleurotus species P. cornucopiae (ATCC 38547) and P. ostreatus (ATCC 34675) were the most efficient.     

Decolourization of Municipal Effluent and Sludge by Pleurotus sajor-caju and Pleurotus ostreatus

Summary The Americana Municipal Treatment Station, S?o Paulo, Brazil, manages 400 l of effluent s⁻¹, from domestic and textile origin, which produces an average of 20 t of sludge per day. The decolourization of the effluent and sludge by three strains of Pleurotus (Pleurotus sajor-caju F2, F6 and Pleurotus ostreatus) was evaluated. The strains of P. sajor-caju F2 and F6 were able to decolourize the sludge, while P. ostreatus was less efficient. Detoxification was appraised with three bioassays comprising the cnidarian Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. After exposure to fungi, effluent toxicity decreased but not that of its sludge. Strain P. sajor-caju F6 presented signs of toxicity shown by electron microscopy in the presence of the effluent. The three strains produced high amounts of manganese-peroxidase (Mn–P) and laccase in the presence of the sludge. Although P. ostreatus produced large amount of Mn–P and laccase enzymes, these enzymes did not result in decolourization of the sludge, suggesting that other factors are likely to be involved. Carbon content decreased only in the treatment with P. ostreatus.     

Two Specific Strains of <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis> causing Mucocutaneous Manifestations of Histoplasmosis: Preliminary Analysis of a Frequent Manifestation of Histoplasmosis in Southern Brazil

Objectives  Skin lesions, uncommon in US cases (<10%), occur in 38–85% of cases reported from Latin America. Although these differences may reflect reporting bias, delayed diagnosis, or differences in host immune response among different ethnic groups, they also could result from genetic differences changing the pathobiology of the organism. It is possible that genetic differences among strains of H. capsulatum may influence the pathogenesis and clinical manifestations of histoplasmosis. Methods  We examined the clinical features of patients with mucocutaneous manifestations of histoplasmosis and performed genetic analysis based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes of H. capsulatum isolates of patients. Two pairs of PCR primers were designed to develop and amplify the ITS regions of H. capsulatum, 5′-TACCCGGCCACCCTTGTCTA-3′ and 5′-AGCGGGTGGCAAAGCCC-3′. These primers were based on the ITS sequence of Ajellomyces capsulatus, the ascomycetous teleomorph form of H. capsulatum, deposited in the GenBank (accession number U18363). Eight patients attending a tertiary-care hospital in southern Brazil were enrolled into the study. All case patients had skin cultures growing H. capsulatum at the mycology laboratory. Results  Six of eight (75%) patients were HIV-positive and presented involvement of multiples organs by H. capsulatum. Two HIV-negative patients did not present evidence of involvement of other organs besides mucosa and skin. ITS sequencing of a DNA H. capsulatum fragment of 485-bp from isolates of 8 patients revealed two distinct strains. The 2 distinct fragments (Hc1, Hc2) differed from each other at 7 positions in the ITS regions. They were identical to strains of H. capsulatum isolated in patients from Colombia and Argentina, but different from strains isolated in US. Hc1 and Hc2 were isolated in 5 patients and 3 patients, respectively, with mucocutaneous manifestations of histoplasmosis. Both Hc1 and Hc2 strains were isolated in HIV-infected and non-HIV-infected patients. Conclusions  Mucocutaneous manifestations of histoplasmosis, which are frequently seen in Brazilian patients were caused by 2 specific strains in our institution. Those strains have been isolated in patients with these particular clinical features of histoplasmosis in Latin America. Our study suggests that unique pathogenic characteristics among the Latin American species of H. capsulatum might explain its increased dermatotropism.