Georgios I. Zervakis

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.

Genetic polymorphism and taxonomic infrastructure of the Pleurotus eryngii species-complex as determined by RAPD analysis, isozyme profiles and ecomorphological characters

The Pleurotus eryngii species-complex includes populations of choice edible mushrooms, growing in the greater Mediterranean area in close association with different genera of plants of the family Apiaceae. Their distinct host-specialization served as the principal criterion for the discrimination of several taxa; however, the genetic relationships among the various P. eryngii ecotypes remain ambiguous. In the present study, 46 Pleurotus strains with a wide range of geographical origins were isolated from Eryngium spp., Ferula communis, Cachrys ferulacea, Thapsia garganica and Elaeoselinum asclepium subsp. asclepium, and were subjected to isozyme and random amplified polymorphic DNA-PCR (RAPD) analysis. The 16 enzyme activities tested were controlled by 28 loci, 11 of which were monomorphic. Host-exclusive zymograms for the Aph (acid phosphatase) and Phe-1 (dopa-phenoloxidase) loci were obtained from Pleurotus strains associated with C. ferulacea. Allele frequencies, genetic diversity and mean diversity were high for isolates from Eryngium spp. and Ferula communis. In RAPD analysis, the use of five primers allowed the production of 45 (out of 48) polymorphic bands, while four molecular markers specific for the identification of Pleurotus strains growing on E. asclepium subsp. asclepium and C. ferulacea were obtained. The Pleurotus strains produced 35 distinct electrophoretic types and 42 RAPD patterns, which independently permitted the separation of the fungal populations into five clusters in accordance with their host-specificity. In addition, the evaluation of the principal ecological and morphological characters provided further evidence for discriminating between P. nebrodensis growing on C. ferulacea and the rest of the host-associated populations. The latter represent taxa at the varietal level: P. eryngii var. eryngii, P. eryngii var. ferulae and P. eryngii var. elaeoselini. The position of taxa of dubious validity, such as P. hadamardii and P. fossulatus, is discussed in relation to the new findings. All Mediterranean Pleurotus populations growing on umbellifers seem to have recently diverged through a sympatric speciation process, that is based on both intrinsic reproductive barriers and extrinsic ecogeographical factors.

Mycelium growth kinetics and optimal temperature conditions for the cultivation of edible mushroom species on lignocellulosic substrates

The influence of environmental parameters on mycelial linear growth ofPleurotus ostreatus, P. eryngii, P. pulmonarius, Agrocybe aegerita, Lentinula edodes, Volvariella volvacea andAuricularia auricula-judae was determined in two different nutrient media in a wide range of temperature, forming the basis for the assessment of their temperature optimaV. volvacea grew faster at 35°C,P. eryngii at 25°C,P. ostreatus andP. pulmonarius at 30°C,A. aegerita at 25 or 30°C andA. auricula-judae at 20 or 25°C depending on the nutrient medium used andL. edodes at 20 or 30°C depending on the strain examined. The mycelium extension rates were evaluated on seven mushroom cultivation substrates: wheat straw, cotton gin-trash, peanut shells, poplar sawdust, oak sawdust, corn cobs and olive press-cake. The mycelium extension rates (linear growth and colonization rates) were determined by the ‘race-tube’ technique, and were found to be the highest on cotton gin-trash, peanut shells and poplar sawdust forPleurotus spp. andA. aegerita. Wheat straw, peanut shells and particularly cotton gin-trash supported fast growth ofV. volvacea, whereas wheat straw was the most suitable substrate forL. edodes andA. auricula-judae. Supplemented oak sawdust and olive press-cake were poor substrates for most species examined, white almost all strains performed adequately on corn cobs.

Edible mushrooms from olive oil mill wastes

Abstract 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.

A pluralistic approach in the study of Pleurotus species with emphasis on compatibility and physiology of the European morphotaxa

The anatomical, physiological, ecological and cultural characters of 11 Pleurotus morphotaxa reported to occur in Europe are described in detail and compared with the outcome of a pairing-analysis performed among monokaryons of 101 dikaryotic strains (including collections from other continents). The results revealed eight intersterility groups in Europe: P. ostreatus, P. pulmonarius, P. cornucopiae, P. eryngii, P. cystidiosus, P. dryinus, P. calyptratus and P. opuntiae . In addition, strains formerly identified as ‘P. sajor-caju’ were found to be intercompatible with P. pulmonarius , while the speciation process currently under way for P. columbinus, P. citrinopileatus and the ecotypes of P. eryngii is discussed. A synthesis of the data obtained so far on Pleurotus systematics is evaluated in the light of recent evidence together with the world distribution of the recognized biological species of the genus.

Antagonistic bacteria of composted agro-industrial residues exhibit antibiosis against soil-borne fungal plant pathogens and protection of tomato plants from Fusarium oxysporum f.sp. radicis-lycopersici

Rhizospheric and root-associated/endophytic (RAE) bacteria were isolated from tomato plants grown in three suppressive compost-based plant growth media derived from the olive mill, winery and Agaricus bisporus production agro-industries. Forty-four (35 rhizospheric and 9 RAE) out of 329 bacterial strains showed in vitro antagonistic activity against at least one of the soil-borne fungal pathogens, Fusarium oxysporum f.sp. radicis-lycopersici (FORL), F. oxysporum f.sp. raphani, Phytophthora cinnamomi, P. nicotianae and Rhizoctonia solani. The high percentage of total isolates showing antagonistic properties (13%) and their common chitinase and β-glucanase activities indicate that the cell wall constituents of yeasts and macrofungi that proliferate in these compost media may have become a substrate that favours the establishment of antagonistic bacteria to soil-borne fungal pathogens. The selected bacterial strains were further evaluated for their suppressiveness to tomato crown and root rot disease caused by FORL. A total of six rhizospheric isolates, related to known members of the genera Bacillus, Lysinibacillus, Enterobacter and Serratia and one RAE associated with Alcaligenes faecalis subsp. were selected, showing statistically significant decrease of plant disease incidence. Inhibitory effects of extracellular products of the most effective rhizospheric biocontrol agent, Enterobacter sp. AR1.22, but not of the RAE Alcaligenes sp. AE1.16 were observed on the growth pattern of FORL. Furthermore, application of cell-free culture extracts, produced by Enterobacter sp. AR1.22, to tomato roots led to plant protection against FORL, indicating a mode of biological control action through antibiosis.

Correlation of the properties of several lignocellulosic substrates to the crop performance of the shiitake mushroom Lentinula edodes

Two selected Lentinula edodes strains (S4080 and SIEF0231) were cultivated on oak-wood sawdust (OS), wheat straw (WS) and corn-cobs (CC) substrates in order to examine the influence of those residues on mycelium growth and on basidiomata production. For both strains, mycelial growth measurements conducted in ‘race tubes’ demonstrated faster colonization of OS and WS media. Lag-phase and complete colonization periods were correlated to mycelium extension rates in the three substrates tested. Similar patterns of pH and electrical conductivity (Ec) changes were detected in all media and for all strains tested; the pH decreased steadily throughout the colonization process to reach values of 4.49–5.06; Ec increased by the end of mycelium colonization, and it presented the highest and lowest values in the WS and OS media respectively. In addition, a negative correlation was established between final salt content of the substrates and mycelium extension rates. Subjecting fully colonized substrates to a cold-shock treatment resulted in fruiting 58–65 days after inoculation in tubes; WS and CC promoted earlier sporophore initiation than OS. Monitoring CO2 emissions by strain SIEF0231 in pilot-scale cultivation on synthetic blocks, revealed higher respiration rates from OS and CC than from WS, which were further correlated with substrate colonization rates. Among residues colonized by the same strain, WS appeared to promote earliness and crop productivity (BE 54.17%) by presenting shorter cropping periods and equal yield distribution among flushes, while on OS and CC maximum yields were obtained within the first two flushes. Moreover, heavier basidiomata were produced by WS and OS substrates.

Genetic Relatedness Among Dioecious Ficus carica L. cultivars by Random Amplified Polymorphic DNA Analysis, and Evaluation of Agronomic and Morphological Characters

A collection of 64 fig (Ficus carica L.) accessions was characterized through the use of RAPD markers, and results were evaluated in conjunction with morphological and agronomical characters, in order to determine the genetic relatedness of genotypes with diverse geographic origin. The results indicate that fig cultivars have a rather narrow genetic base. Nevertheless, RAPD markers could detect enough polymorphism to differentiate even closely related genotypes (i.e., clones of the same cultivar) and a unique fingerprint for each of the genotypes studied was obtained. No wasteful duplications were found in the collection. Cluster analysis allowed the identification of groups in accordance with geographic origin, phenotypic data and pedigree. Taking into account the limited information concerning fig cultivar development, the results of this study, which provide information on the genetic relationships of genetically distinct material, dramatically increase the fundamental and practical value of the collection and represent an invaluable tool for fig germplasm management.

Bacterial diversity in spent mushroom compost assessed by amplified rDNA restriction analysis and sequencing of cultivated isolates.

Spent mushroom compost (SMC) is the residual by-product of commercial Agaricus spp. cultivation, and it is mainly composed of a thermally treated cereal straw/animal manure mixture colonized by the fungal biomass. Research on the valorization of this material is mainly focusing on its use as soil conditioner and plant fertilizer. An investigation of the bacterial diversity in SMC was performed using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils. The bacterial population was estimated by the plate count method to a mean of 2.7 10(9) colony forming units (cfu) per g of dry weight, while the numbers of Gram-positive and Gram-negative bacteria were 1.9 10(9) and 4.9 10(8) cfu per g dw respectively as estimated by enumeration on semi-selective media. Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene. Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera Bacillus, Paenibacillus, Exiguobacterium, Staphylococcus, Desemzia, Carnobacterium, Brevibacterium, Arthrobacter and Microbacterium of the bacterial divisions Firmicutes and Actinobacteria. Two bacterial groups have phylogenetic links with the genera Comamonas and Sphingobacterium, which belong to beta-Proteobacteria and Bacteroidetes respectively. Two potentially novel bacteria are reported, which are associated with the genera Bacillus and Microbacterium. Most of the bacteria identified are of environmental origin, while strains related to species usually isolated from insects, animal and clinical sources were also detected. It appears that bacterial diversity in SMC is greatly affected by the origin of the initial material, its thermal pasteurization treatment and the potential unintended colonization of the mushroom substrate during the cultivation process.

Genetic variability and systematics of eleven Pleurotus species based on isozyme analysis

Eleven enzyme activities were used to study the intraspecific diversity and interspecific relatedness of 11 Pleurotus species: P. abalonus, P. columbinus, P. cornucopiae, P. cystidiosus, P. dryinus, P. eryngii, P. flabellatus, P. ostreatus, P. pulmonarius, P. sajor-caju, and P. sapidus. In addition, the zymograms of their homokaryotic progenies were evaluated to determine the number of genes and alleles encoding them. Among the 18 loci detected, three coded for PEP A, two for G6PD, LAP, MDH, PEP C and PGM, and one for APH, HK, IDH, 6PGD and PHI. Based on electrophoretic patterns, the 77 wild Pleurotus dikaryons were divided into 51 genotypic classes. The higher intraspecific genetic variation was demonstrated by P. cystidiosus and P. eryngii, while P. cornucopiae, P. dryinus and P. sajor-caju appeared to be the least diverse species examined. Furthermore, with the aid of numerical analysis, valid species such as P. cornucopiae, P. dryinus, P. eryngii and P. flabellatus were clearly distinguished. Among the rest of the taxa studied, the derived dendrograms permitted the delimitation between P. abalonus-P. cystidiosus and P. columbinus-P. ostreatus, although the former members of these two pairs were previously considered as varieties. On the other hand P. pulmonarius and P. sajor-caju formed a tight cluster indicative of a certain genetic affinity, which questions their original designation as distinct species. As regards P. sapidus, it presented a slightly higher proximity to the P. pulmonarius-P. sajor-caju complex than to P. ostreatus. Finally, the phylogenetic implications from the findings of this work are discussed.