Tecnologic Development on Pleurotus Cultivation: Specific Practices Used in Brazil

HIGHLIGHTS P. ostreatus and P. sapidus are the most productive species under the evaluated conditions. Different growing systems are suitable for the production of P. ostreatus var. Florida. Temperature control level affects differently the P. ostreatus var. Florida isolates. Environmental and strain factors affect yield and production parameters of P. ostreatus var. Florida.

Abstract In Brazil, Pleurotus is the most important mushroom produced especially P. ostreatus var. Florida. In this country as in many others, the great potential for mushroom cultivation remains unexplored. Therefore, it is very important to develop new studies that allow optimizing its production. The aims of the manuscript were: i) to evaluate the productivity of six different species of Pleurotus (P. citrinopileatus; P. djamor; P. ostreatus; P. ostreatus var. Florida; P. pulmonarius; P. sapidus); ii) to measure the effect of three different environmental conditions during cultivation of three isolates of P. ostreatus var. Florida. As results, P. ostreatus and P. sapidus were the most productive isolates under the evaluated conditions. Different environments produced variable effects according to the P. ostreatus var. Florida isolates, being possible to observe a highly plastic strain (POF 02/18), a highly sensitive strain (POF 03/18) and a strain with variable responses (POF 01/18).

Promising cellulolytic fungi isolates for rice straw degradation.

The objective of this study was to evaluate the potential of eight fungal isolates obtained from soils in rice crops for straw degradation in situ. From the initial eight isolates, Pleurotus ostreatus T1.1 and Penicillium sp. HC1 were selected for further characterization based on qualitative cellulolytic enzyme production and capacity to use rice straw as a sole carbon source. Subsequently, cellulolytic, xylanolytic, and lignolytic (Pleurotus ostreatus) activity on carboxymethyl cellulose, oat xylan, and rice straw with different nitrogen sources was evaluated. From the results obtained it was concluded both isolates are capable to produce enzymes necessary for rice straw degradation. However, their production is dependent upon carbon and nitrogen source. Last, it was established that Pleurotus ostreatus T1.1 and Penicillium sp. HC1 capability to colonize and mineralize rice straw, in mono-and co-culture, without affecting nitrogen soil content.

Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white-rot agaricomycete Pleurotus ostreatus.

White-rot fungi play an important role in the global carbon cycle because they are the species that almost exclusively biodegrade wood lignin in nature. Lignin peroxidases (LiPs), manganese peroxidases (MnPs) and versatile peroxidases (VPs) are considered key players in the ligninolytic system. Apart from LiPs, MnPs and VPs, however, only few other factors involved in the ligninolytic system have been investigated using molecular genetics, implying the existence of unidentified elements. By combining classical genetic techniques with next-generation sequencing technology, they successfully showed an efficient forward genetics approach to identify mutations causing defects in the ligninolytic system of the white-rot fungus Pleurotus ostreatus. In this study, they identified two genes - chd1 and wtr1 - mutations in which cause an almost complete loss of Mn2+ -dependent peroxidase activity. The chd1 gene encodes a putative chromatin modifier, and wtr1 encodes an agaricomycete-specific protein with a putative DNA-binding domain. The chd1-1 mutation and targeted disruption of wtr1 hamper the ability of P. ostreatus to biodegrade wood lignin. Examination of the effects of the aforementioned mutation and disruption on the expression of certain MnP/VP genes suggests that a complex mechanism underlies the ligninolytic system in P. ostreatus.

Morphological and molecular characterization of yellow oyster mushroom, Pleurotus citrinopileatus, hybrids obtained by interspecies mating.

Pleurotus citrinopileatus (yellow oyster mushroom) has an attractive shape and yellow colour but the fragile texture complicates packaging, and its strong aroma is unappealing to consumers. This study aimed to improve the characteristics and yield of P. citrinopileatus by interspecies mating between monokaryotic cultures of P. citrinopileatus and P. pulmonarius. Ten monokaryon cultures of the parental lines were crossed in all combinations to obtain hybrids. Eleven compatible mating pairs were obtained and cultivated to observe their sporophore morphology and yield. The selected hybrid, i.e. P1xC9, was beige in colour while hybrid P3xC8 was yellow in colour. Their sporophores had less offensive aroma, improved texture and higher yield. The DNA sequences of these hybrids were found to be in the same clade as the P. citrinopileatus parent with a bootstrap value of 99%. High bootstrap values indicate high genetic homology between hybrids and the P. citrinopileatus parent. The biological efficiencies of these hybrids P1xC9 (70.97%) and P3xC8 (52.14%) were also higher than the P. citrinopileatus parent (35.63%). Interspecies hybrids obtained by this mating technique can lead to better strains of mushrooms for genetic improvement of the Pleurotus species.

Long term storage of Pleurotus ostreatus and Trametes versicolor isolates using different cryopreservation techniques and its impact on laccase activity.

The strain Pleurotus ostreatus Florida f6, its 45 basidiospore-derived isolates (both monokaryons and dikaryons prepared in our laboratory), Trametes versicolor strain CCBAS 614 and 22 other T. versicolor isolates obtained from the sporocarps collected in distant localities were successfully preserved for 12 y using perlite and straw cryopreservation protocols. All tested isolates survived a 12-year storage in liquid nitrogen (LN) and their laccase production and Poly B411 decolorization capacity was preserved. Also mycelium extension rate and the types of colony appearance of individual isolates remained unchanged. Different cryopreservation techniques were also tested for the short time (24 h) and the long time (6 m) storage of the culture liquid with extracellular laccase produced by T. versicolor strain CCBAS 614. The results showed that 10 % glycerol was the most suitable cryopreservant. The absence of the cryopreservant did not cause high loss of laccase activity in the samples; the presence of DMSO (5 or 10 %) in LN-stored samples caused mostly a decrease of laccase activity. For the preservation of laccase activity in the liquid culture the storage in the freezer at -80 °C is more convenient than the storage in liquid nitrogen.

Capacity of a newly isolated fungus Pleurotus eryngii from Tunceli, Ovacik for chemical oxygen demand reduction and biodecolorization of Azo-Dye Congo Red.

Biodecolorization of Congo red dye in both agar—plate and agitated liquid culture mediums by newly isolated white rot fungus Pleurotus eryngii has been studied. This fungus isolated from Tunceli—Ovacik province of Turkey. We have also examined the chemical oxygen demand reduction after decolorization under agitated liquid culture medium. For agar plate screening the decolorization capacity of P. eryngii, growth and decolorization halos were determined on saboroud dextrose agar (SDA) plates containing 0.05, 0.1, 0.5, 1 and 2 g/l of Congo red. P. eryngii showed certain decolorization capacities and was able to decolorize all studied concentrations of Congo red, but not to the same extent. Our results indicated that the new isolate P. eryngii had maximum decolorization (87% at 100 mg/l initial dye concentration) and chemical oxygen demand reduction (82% at 25 mg/l initial dye concentration) activities after 7 days under agitated submerged culture conditions. This new isolate could be an effective bioremediation tool for treatment of Congo red containing textile wastewater.

Genetic diversity of Kenyan native oyster mushroom (Pleurotus).

Members of the genus Pleurotus, also commonly known as oyster mushroom, are well known for their socioeconomic and biotechnological potentials. Despite being one of the most important edible fungi, the scarce information about the genetic diversity of the species in natural populations has limited their sustainable utilization. A total of 71 isolates of Pleurotus species were collected from three natural populations: 25 isolates were obtained from Kakamega forest, 34 isolates from Arabuko Sokoke forest and 12 isolates from Mount Kenya forest. Amplified fragment length polymorphism (AFLP) was applied to thirteen isolates of locally grown Pleurotus species obtained from laboratory samples using five primer pair combinations. AFLP markers and internal transcribed spacer (ITS) sequences of the ribosomal DNA were used to estimate the genetic diversity and evaluate phylogenetic relationships, respectively, among and within populations. The five primer pair combinations generated 293 polymorphic loci across the 84 isolates. The mean genetic diversity among the populations was 0.25 with the population from Arabuko Sokoke having higher (0.27) diversity estimates compared to Mount Kenya population (0.24). Diversity between the isolates from the natural population (0.25) and commercial cultivars (0.24) did not differ significantly. However, diversity was greater within (89%; P > 0.001) populations than among populations. Homology search analysis against the GenBank database using 16 rDNA ITS sequences randomly selected from the two clades of AFLP dendrogram revealed three mushroom species: P. djamor, P. floridanus and P. sapidus; the three mushrooms form part of the diversity of Pleurotus species in Kenya. The broad diversity within the Kenyan Pleurotus species suggests the possibility of obtaining native strains suitable for commercial cultivation.

Estudo do potencial anti-leishmania e anti-trypanosoma cruzi do ergosterol isolado do basidiomiceto pleurotus salmoneostramineus

Considerando a necessidade de novos tratamentos para doenças negligenciadas como a leishmaniose visceral e a doença de Chagas, o presente trabalho realizou o fracionamento do basidiomiceto comestível Pleurotus salmoneos tramineus na busca por substâncias potencialmente antiparasitárias. Dentre as frações ativas, foi isolado um composto denoninado ergosterol, o qual apresentou atividade anti-Leishmania (L.) infantum e anti-Trypanosoma cruzi. O ergosterol foi ativo contra amastigotas intracelulares de Leishmania (L.) infantum, com valor de Concentração Efetiva 50% (CE50) de 125 µM e de 129 µM contra formas tripomastigotas de Trypanosoma cruzi. O estudo da citotoxicidade em células de mamífero resultou em um valor de CE50 de 619 µM. Seu mecanismo de ação em tripomastigotas resultou uma rápida permeabilização da membrana plasmática, com a despolarização do potencial de membrana mitocondrial,levando o parasito à morte. Apesar disso, não se verificou aumento de espécies reativas de oxigênio no parasito, demonstrando que seu mecanismo de ação não envolve a indução de estresse oxidativo. A seleçãode metabólitos secundários antiparasitários presentes na natureza podefornecer futuros protótipos para o desenho de novos fármacos para doenças negligenciadas.

Considering the need for new treatments for neglected diseases as visceralleishmaniasis and Chagas disease, in this work we fractionated the ediblemushroom Pleurotus salmoneostramineus in the search for potentialantiparasitic compounds. Among the active fractions, it was isolated theergosterol, which showed anti-Leishmania (L.) infantum e anti-Trypanosomacruzi activities. The ergosterol was active against intracellular amastigotes ofLeishmania (L.) infantum and trypomastigotes of Trypanosoma cruzi, with50% Inhibitory Concentration (IC50) values of 125 µM and 129 µM,respectively. The cytotoxicity in mammalian cells resulted in an IC50 value of619 µM. Its mechanism of action in Trypanosoma cruzi trypomastigotesresulted in permeabilization of the plasma membrane, as well asdepolarization of mitochondrial membrane potential, leading to parasitedeath. Nevertheless, there was no increase in reactive oxygen species,demonstrating that its mechanism of action does not involve the induction ofoxidative stress in the parasite. The selection of antiparasitic secondarymetabolites present in nature can provide future prototypes for the design ofnew drugs for neglected diseases.

Morphology and mycelial growth rate of Pleurotus spp. strains from the Mexican mixtec region.

Two native Pleurotus spp. strains (white LB-050 and pale pink LB-051) were isolated from rotten tree trunks of cazahuate (Ipomoea murucoides) from the Mexican Mixtec Region. Both strains were chemically dedikaryotized to obtain their symmetrical monokaryotic components (neohaplonts). This was achieved employing homogenization time periods from 60 to 65 s, and 3 day incubation at 28 °C in a peptone-glucose solution (PGS). Pairing of compatible neohaplonts resulted in 56 hybrid strains which were classified into the four following hybrid types: (R(1-n)xB(1-n), R(1-n)xB(2-1), R(2-n)xB(1-n) and R(2-n)xB(2-1)). The mycelial growth of Pleurotus spp. monokaryotic and dikaryotic strains showed differences in texture (cottony or floccose), growth (scarce, regular or abundant), density (high, regular or low), and pigmentation (off-white, white or pale pink). To determine the rate and the amount of mycelium growth in malt extract agar at 28 °C, the diameter of the colony was measured every 24 h until the Petri dish was completely colonized. A linear model had the best fit to the mycelial growth kinetics. A direct relationship between mycelial morphology and growth rate was observed. Cottony mycelium presented significantly higher growth rates (p < 0.01) in comparison with floccose mycelium. Thus, mycelial morphology can be used as criterion to select which pairs must be used for optimizing compatible-mating studies. Hybrids resulting from cottony neohaplonts maintained the characteristically high growth rates of their parental strains with the hybrid R(1-n)xB(1-n) being faster than the latter.

Characterization of interspecific hybrid dikaryons of the oyster mushrooms, Pleurotus florida PAU-5 and P. sajor-caju PAU-3 (higher Basidiomycetes) from India.

Five Pleurotus hybrid dikaryons, developed through cross-breeding of P. florida PAU-5 (PF-5) and P. sajor-caju PAU-3 (PSC-3) were characterized with respect to textural properties, color, and enzymatic and genetic variability. Texture profile revealed significant differences in springiness, resilience, cohesiveness, and chewiness between all hybrids compared to the parents. Among the hybrid cultures, maximum whiteness was reported in hybrid 37, whereas hybrid 8 had minimum whiteness. Three hybrids (16, 37, 42) showed an increased linear growth rate in relation to PF-5, whereas no hybrid showed a higher growth rate than PSC-3. Maximum endoglucanase and xylanase activity was observed in hybrid 46, whereas minimum activity occurred in hybrid 42. Laccase and protease activity was higher in hybrid 37 and 46, respectively. Four hybrids (16, 37, 42, 46) showed increased peroxidase activity in relation to PF-5, whereas hybrid 46 showed activity higher than the parent PSC-3. Comparison of isozyme patterns confirmed the hybrid nature of hybrid 16. The large variation in the intensity of bands could be a result of recombination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of extracellular enzymes revealed 60.3- and 43-KDa bands in all the hybrids. An additional 25-KDa band was reported in hybrids 37, 42, and 46 and the parent PF-5, indicating their close relatedness. Parental strains showed higher divergence in small-subunit ribosomal DNA region compared with the internal transcribed spacer region, indicating their significance in varietal discrimination. Hybrid 46 had a small-subunit ribosomal DNA region more similar to that of PSC-3 compared with PF-5, whereas the internal transcribed spacer region of hybrids 42 and 46 revealed close resemblance to that of PF-5 and PSC-3, respectively.

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.

Morphology and mycelial growth rate of Pleurotus spp. strains from the Mexican mixtec region

Two native Pleurotus spp. strains (white LB-050 and pale pink LB-051) were isolated from rotten tree trunks of cazahuate (Ipomoea murucoides) from the Mexican Mixtec Region. Both strains were chemically dedikaryotized to obtain their symmetrical monokaryotic components (neohaplonts). This was achieved employing homogenization time periods from 60 to 65 s, and 3 day incubation at 28 °C in a peptone-glucose solution (PGS). Pairing of compatible neohaplonts resulted in 56 hybrid strains which were classified into the four following hybrid types: (R1-n xB1-n, R1-n xB2-1, R2-n xB1-n and R2-n xB2-1). The mycelial growth of Pleurotus spp. monokaryotic and dikaryotic strains showed differences in texture (cottony or floccose), growth (scarce, regular or abundant), density (high, regular or low), and pigmentation (off-white, white or pale pink). To determine the rate and the amount of mycelium growth in malt extract agar at 28 °C, the diameter of the colony was measured every 24 h until the Petri dish was completely colonized. A linear model had the best fit to the mycelial growth kinetics. A direct relationship between mycelial morphology and growth rate was observed. Cottony mycelium presented significantly higher growth rates (p < 0.01) in comparison with floccose mycelium. Thus, mycelial morphology can be used as criterion to select which pairs must be used for optimizing compatible-mating studies. Hybrids resulting from cottony neohaplonts maintained the characteristically high growth rates of their parental strains with the hybrid R1-n xB1-n being faster than the latter.

Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants.

Aflatoxin B1 (AFB1) is a highly toxic fungal metabolite having carcinogenic, mutagenic and teratogenic effects on human and animal health. Accidental feeding of aflatoxin-contaminated rice straw may be detrimental for ruminant livestock and can lead to transmission of this toxin or its metabolites into the milk of dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus produces ligninolytic enzymes like laccase and manganese peroxidase (MnP). These extracellular enzymes have been reported to degrade many environmentally hazardous compounds. The present study examines the ability of P. ostreatus strains to degrade AFB1 in rice straw in the presence of metal salts and surfactants. Laccase and MnP activities were determined spectrophotometrically. The efficiency of AFB1 degradation was evaluated by high performance liquid chromatography. Highest degradation was recorded for both P. ostreatus MTCC 142 (89.14 %) and P. ostreatus GHBBF10 (91.76 %) at 0.5 µg mL(-1) initial concentration of AFB1. Enhanced degradation was noted for P. ostreatus MTCC 142 in the presence of Cu(2+) and Triton X-100, at toxin concentration of 5 µg mL(-1). P. ostreatus GHBBF10 showed highest degradation in the presence of Zn(2+) and Tween 80. Liquid chromatography-mass spectrometric analysis revealed the formation of hydrated, decarbonylated and O-dealkylated products. The present findings suggested that supplementation of AFB1-contaminated rice straw by certain metal salts and surfactants can improve the enzymatic degradation of this mycotoxin by P. ostreatus strains.

Extracellular polysaccharide production by a strain of Pleurotus djamor isolated in the south of Brazil and antitumor activity on Sarcoma 180

Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072) and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24) and maximal biomass productivity (Q Xmax = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

Extracellular polysaccharide production by a strain of Pleurotus djamor isolated in the south of Brazil and antitumor activity on Sarcoma 180.

Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (YP/S = 0.072) and maximum extracellular polysaccharide productivity (Q(Pmax) = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (YX/S = 0.24) and maximal biomass productivity (Q(Xmax) = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm(3) in untreated group and 1.6 cm(3) in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

Secretion of laccase and manganese peroxidase by Pleurotus strains cultivate in solid-state using Pinus spp. sawdust

Pleurotus species secrete phenol oxidase enzymes: laccase (Lcc) and manganese peroxidase (MnP). New genotypes of these species show potential to be used in processes aiming at the degradation of phenolic compounds, polycyclic aromatic hydrocarbons and dyes. Hence, a screening of some strains of Pleurotus towards Lcc and MnP production was performed in this work. Ten strains were grown through solid-state fermentation on a medium based on Pinus spp. sawdust, wheat bran and calcium carbonate. High Lcc and MnP activities were found with these strains. Highest Lcc activity, 741 ± 245 U gdm-1 of solid state-cultivation medium, was detected on strain IB11 after 32 days, while the highest MnP activity occurred with strains IB05, IB09, and IB11 (5,333 ± 357; 4,701 ± 652; 5,999 ± 1,078 U gdm-1, respectively). The results obtained here highlight the importance of further experiments with lignocellulolytic enzymes present in different strains of Pleurotus species. Such results also indicate the possibility of selecting more valuable strains for future biotechnological applications, in soil bioremediation and biological biomass pre-treatment in biofuels production, for instance, as well as obtaining value-added products from mushrooms, like phenol oxidase enzymes.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

Pleurotus eryngii species complex: sequence analysis and phylogeny based on partial EF1α and RPB2 genes.

The Pleurotus eryngii species complex comprises at least six varieties (var. eryngii (DC.: Fr) Quel., ferulae Lanzi, elaeoselini Venturella et al., nebrodensis (Inzenga) Sacc., tingitanus Lewinsohn et al. and tuoliensis C.J. Mou). This species is unique among the genus Pleurotus because in nature it is found in association with certain species of the Apiaceae (Umbelliferae) and Asteraceae (Compositae) families. Sequences of partial regions of the translation elongation factor (EF1α) and RNA polymerase II (RPB2) genes were analyzed in order to detect nucleotide polymorphisms that might unequivocally distinguish varieties eryngii, ferulae, elaeoselini and nebrodensis. A phylogenetic analysis was also performed with an aim to establish phylogenetic relationships among those. Sequence analysis of the partial EF1α and RPB2 genes contained nucleotide polymorphisms able to unequivocally distinguish variety nebrodensis from the rest. However, distinction among eryngii, elaeoselini and ferulae was achieved only through the RPB2 gene. The phylogenetic analyses from the combined data sets (EF1α and RPB2) indicated that P. eryngii is a monophyletic group and that varieties eryngii, elaeoselini and ferulae are closely related. P. eryngii var. nebrodensis was placed in a distinct clade clearly differentiated from the other varieties but still monophyletic with the P. eryngii complex. The limited nucleotide variation in partial EF1α and RPB2 among varieties eryngii, ferulae and elaeoselini supports the placement of these groups as varieties and not species within the complex.

Molecular identification of Trichoderma species associated with Pleurotus ostreatus and natural substrates of the oyster mushroom.

Green mold of Pleurotus ostreatus, caused by Trichoderma species, has recently resulted in crop losses worldwide. Therefore, there is an emerging need for rapid means of diagnosing the causal agents. A PCR assay was developed for rapid detection of Trichoderma pleurotum and Trichoderma pleuroticola, the two pathogens causing green mold of P. ostreatus. Three oligonucleotide primers were designed for identifying these species in a multiplex PCR assay based on DNA sequences within the fourth and fifth introns in the translation elongation factor 1alpha gene. The primers detected the presence of T. pleurotum and/or T. pleuroticola directly in the growing substrates of oyster mushrooms, without the need for isolating the pathogens. The assay was used to assess the presence of the two species in natural environments in which P. ostreatus can be found in Hungary, and demonstrated that T. pleuroticola was present in the growing substrates and on the surface of the basidiomes of wild oyster mushrooms. Other Trichoderma species detected in these substrates and habitats were Trichoderma harzianum, Trichoderma longibrachiatum and Trichoderma atroviride. Trichoderma pleurotum was not found in any of the samples from the forested areas tested in this study.