Use of molecular markers for differentiation of cultivated strains of oyster and button mushrooms

Analysis of commercial strains of two edible mushrooms, Pleurotus ostreatus and Agaricus bisporus, using PCR and isozyme electrophoresis techniques allowed us to differentiate groups of genetically similar and distant strains. Among the commercial strains of P. ostreatus, the level of genetic variation was higher suggesting a broader genetic basis employed in breeding of this mushroom. The cultivars and hybrids of, A. bisporus, showed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.     

Cultivation of Pleurotus ostreatus and other edible mushrooms

Pleurotus ostreatus is the second most cultivated edible mushroom worldwide after Agaricus bisporus. It has economic and ecological values and medicinal properties. Mushroom culture has moved toward diversification with the production of other mushrooms. Edible mushrooms are able to colonize and degrade a large variety of lignocellulosic substrates and other wastes which are produced primarily through the activities of the agricultural, forest, and food-processing industries. Particularly, P. ostreatus requires a shorter growth time in comparison to other edible mushrooms. The substrate used for their cultivation does not require sterilization, only pasteurization, which is less expensive. Growing oyster mushrooms convert a high percentage of the substrate to fruiting bodies, increasing profitability. P. ostreatus demands few environmental controls, and their fruiting bodies are not often attacked by diseases and pests, and they can be cultivated in a simple and cheap way. All this makes P. ostreatus cultivation an excellent alternative for production of mushrooms when compared to other mushrooms.     

Mining and utilization of mushroom ESTs for microsatellites

In the present study we report on the exploitation of expressed sequence tags (ESTs); (1) to investigate whether microsatellite densities are significantly differed among Pleurotus ostreatus, Lentinula edodes and Agaricus bisporus ESTs, (2) between development stages of mycelia and fruiting bodies and (3) to identify microsatellite primer pairs that could be used in mushroom genetic studies. Analyses of ESTs indicated that three mushroom species and tissues showed statistically significant microsatellite densities. A total of 23 EST-microsatellite primer pairs were developed and tested on two species of mushrooms. The use of these microsatellite primer pairs could be used in genetic studies of mushroom species.     

Behavioural response of the fungus gnat,Bradysia impatiens (Diptera: Sciaridae) towards certain edible mushrooms and saprophytic fungi

The fungus gnat Bradysia impatiens (Johannsen) is a worldwide pest inhabiting organic-rich environments, including mushroom-cultivation substrates, which leads to massive production losses of edible mushrooms. To promote a non-insecticidal pest control strategy, we evaluated adult behavioural response towards two saprophytic fungal strains Aspergillus flavus Link and Mucor circinelloides Tiegh., isolated from B. impatiens-inhabiting substrates and six edible mushrooms species. Our results indicated that (a) B. impatiens was most attracted to the oyster mushroom Pleurotus ostreatus among all six edible mushrooms; (b) males and females demonstrated a significant attraction for M. circinelloides over A. flavus and P. ostreatus; (c) adults demonstrated dynamic attraction tendencies that varied with the culture periods of A. flavus and M. circinelloides, wherein females represented most (mean = 78.6%) of the attracted individuals; and (d) larvae that fed on mycelial cultures of M. circinelloides could complete their entire life cycles, but larvae could not survive on a non-fungal or A. flavus diet. These results demonstrate the potential for the development of M. circinelloides in a push-pull strategy to biocontrol this pest in edible mushroom cultivation.     

Resistance to and uptake of heavy metals in mushrooms

Resistance levels to heavy metals in 21 mushrooms obtained mainly from Japan, but also from Thailand, were tested. The mushrooms were 7 Pleurotus species, 2 Pycnoporus and Pholiota species and one each of Agrocybe, Cryptoporus, Coriolus, Inonotus, Lampteromyces, Grifola, Flammulina, Lyophyllum, Agaricus, and Polyporus species. The Pleurotus species strains showed higher resistance to the heavy metals, copper, cadmium, zinc, nickel, cobalt, mercury than the other species. Pleurotus ostreatus exhibited the highest resistance to all these heavy metals. Pholiota species, Flammulina veltipes, Lyophyllum ulmarium, Agaricus bisporus and Polyporus arcularius were rather sensitive to all the metals tested. The accumulation of copper, zinc and cadmium in Pleurotus ostreatus was studied. The uptake of heavy metals into the mycelia of P. ostreatus increased proportionally to an increasing concentration of these metals in the medium.     

Isolation of a gram-positive bacterium effective in suppression of brown blotch disease of cultivated mushrooms,Pleurotus ostreatus andAgaricus bisporus, caused byPseudomonas tolaasii

A Gram-positive bacterium was isolated from a rottingPleurotus ostreatus fruiting body that markedly reduced the level of extracellular toxins (i.e., tolaasins) produced byPseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The isolated bacterium is saprophytic but not parasitic nor pathogenic toP. ostreatus. A low ratio, ca. 10⁻³ cells of the isolated bacterium for oneP. tolaasii cells, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease inP. ostreatus andAgaricus bisporus. The suppression of the disease development, however requires the initial cell density equivalent to ca. 10⁻¹ cells of the isolated bacterium for one cells of the pathogen. The effects is ascribed to the inactivation of tolaasin by the live, suppressive bacterial cells, and not to metabolites secreted from the organism into culture media. Examination by conventional bacteriological tests and with testing kits, i.e., MicroStation^TMSystem Release 3.5 (Biolog Inc., Hayward, CA), ATB Expression (bioMerieux Inc. Japan) and VITEK (bioMerieux Inc. Japan), failed to assign the organism to any defined bacterial genus. The suppressive bacterium may be useful in future for the development of biocontrol system and/or the construction of genetically modified edible fungi resistant to the disease caused byP. tolaasii.     

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes

Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.     

Pressurized water extraction of β‐glucan enriched fractions with bile acids‐binding capacities obtained from edible mushrooms

A pressurized water extraction (PWE) method was developed in order to extract β‐glucans with bile acids‐binding capacities from cultivated mushrooms (Agaricus bisporus, Lentinula edodes, and Pleurotus ostreatus) to be used as supplements to design novel foods with hypocholesterolemic properties. Extraction yields were higher in individual than sequential extractions being the optimal extraction parameters: 200°C, 5 cycles of 5 min each at 10.3 MPa. The crude polysaccharide (PSC) fractions, isolated from the PWE extracts contained mainly β‐glucans (including chitooligosaccharides deriving from chitin hydrolysis), α‐glucans, and other PSCs (hetero‐/proteo‐glucans) depending on the extraction temperature and mushroom strain considered. The observed bile acids‐binding capacities of some extracts were similar to a β‐glucan enriched fraction obtained from cereals. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:391–400, 2014     

Valorization of solid olive mill wastes by cultivation of a local strain of edible mushrooms

Olive oil industry generates huge quantities of solid olive mill wastes (SOMW), causing environmental damage. Cultivation of edible mushrooms, such as Pleurotus ostreatus is a valuable approach for SOMW valorization. A local strain mycelium (Tizi-Ouzou, Algeria) of P. ostreatus (LPO) was isolated from castor oil plants. Oyster mushroom spawn, produced on barley grains, was used to inoculate wet SOMW, steamed in a traditional steamer during 45 min. The mycelium growth rate on SOMW was first estimated in Petri dish by measuring the surface colonized by the mycelium. The fruit body yields were estimated on culture bags containing 2 kg each of SOMW inoculated at 7% (w/w). The local strain potential was compared with that of a commercial one. Both strains produced high-quality mushrooms, but with low yields. The supplementation of the SOMW with wheat straw at the rate of 10% and 2% of CaCO₃ had significantly enhanced the productivity of the two strains, multiplying it by 3.2 for LPO and by 2.6 for CPO.     

Protein enrichment and digestibility of soft rush (<Emphasis Type="Italic">Juncus effusus</Emphasis>) and rice residues using edible mushrooms <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus species are found to be among the most efficient lignocellulolytic types of white-rot fungi. Rice is the main grain cultivated in the extreme south of Brazil. Defatted rice bran and straw are by-products of low aggregate value. Soft rush (Juncus effusus) is a common native plant also very abundant in the region. In the present work, we evaluated changes in substrate composition after growth of two white-rot fungal species: Pleurotus ostreatus and Pleurotus sajor-caju, aiming to increase protein content and digestibility from substrates through solid fermentations and obtain edible mushrooms of high aggregate value. For that, defatted rice bran, defatted rice straw and soft rush were utilized as substrate. The influence of the variables thermal treatment temperature of substrate, substrate moisture and concentration were evaluated on the protein content, digestibility and biological efficiency. The highest protein enrichment of rice bran in P. sajor-caju-fermented medium was due the fact that there was no fructification in these media, while for the P. ostreatus-fermented medium, part of the synthesized protein was converted into mushrooms. The highest protein enrichments were verified in medium with 80% moisture and 25% soft rush (47.1% using P. ostreatus and 49.0% using P. sajor-caju). A higher digestible protein increase was obtained for both species in media with 70% moisture and 25% soft rush.     

RAPD molecular differentiation of the cultivated strains of the jelly mushrooms, <Emphasis Type="Italic">Auricularia auricula</Emphasis> and <Emphasis Type="Italic">A. polytricha</Emphasis>

Auricularia mushrooms are the fourth most important cultivated mushrooms in the world, with a unique jelly taste and horizontal-septated basidium which are significantly different from other cultivated mushrooms. Differentiation of commercial cultivated strains is difficult to conduct, due to the lack of useful distinguishable characters. In this study, we used the RAPD technique to differentiate 11 commercial strains of A. auricula and five commercial strains of A. polytricha and one white-fruitbody mutant strain, and to characterize their genetic diversity. Results showed that all the strains tested could be differentiated by pooled RAPD data, and even one individual primer (S10) could also discriminate all tested strains. RAPD analysis could differentiate strains having identical rDNA RFLP and supports the classification of the white-fruitbody mutant to the species of A. polytricha. Genetic similarity analysis and grouping derived from RAPD markers reveals a high level of genetic diversity of commercial strains of Auricularia auricula and A. polytricha. Therefore, the RAPD technique can provide a powerful tool to discriminate the commercialAuricularia strains and offer the molecular information useful for breeding systems.     

Genetic Linkage Map of the Edible Basidiomycete Pleurotus ostreatus

We have constructed a genetic linkage map of the edible basidiomycete Pleurotus ostreatus (var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (est1); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of P. ostreatus, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of P. ostreatus.     

Assessment of genetic relatedness among commercial and wild strains of Agaricus bisporus using repetitive extragenic palindromic sequences and polymerase chain reaction

The genetic relatedness among 18 strains of Agaricus bisporus was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by BOX, ERIC (ERICIR-I/ERIC2) and REP (REP1RI/REP2I) gene sequences. Based on the banding patterns of PCR-amplified products, eight putative groups among the 18 commercial and wild strains were recognized. REP-PCR generated multiple distinct products showing considerable variability among the strains with ERIC and REP elements successfully enabled detection of wild and commercial A. bisporus. Strains originating from the same geographical location were not always genetically related. To our knowledge, this was the first relevance study of biodiversity in commercial and native populations of A. bisporus by using the REP-PCR technique. The results confirmed the usefulness REP-PCR typing in intraspecific genetic variation assessments of the button mushroom. High level of Iranian wild strains distance with the commercial cultivars approves their importance as a promising new source of diversity in A. bisporus breeding program.     

Evaluation of genetic diversity of Chinese Pleurotus ostreatus cultivars using DNA sequencing technology

Pleurotus spp. are well-known and economically important cultivated mushrooms in China. Knowledge of the genetic relationship between the Chinese cultivars is essential to the improvement of P. ostreatus strains. Sequence analysis of the internal transcribed spacers (ITS), translation elongation factor (EF1α) and the second largest subunit of RNA polymerase II (RPB2) was performed to assess the genetic diversity of Pleurotus ostreatus strains cultivated in China. The phylogenetic tree constructed using the combined results of the ITS, EF1α and RPB2 sequence analyses showed the genetic relationships between the studied strains. Our phylogenetic analyses therefore provided valuable information on the relationships among the P. ostreatus strains used in this study and that was useful for examining genetic diversity among these strains.     

Genetic diversity,core collection and breeding history of Pleurotus ostreatus in China

Pleurotus ostreatus is one of the most widespread and favourably cultivated mushrooms in China. The cultivated strains of this species have been frequently exchanged domestically and internationally, but no detailed breeding history has been documented. The frequent domestic and international exchange of strains combined with a non-detailed historical documentation of breeding might have led to confusion about strain names and genetic background. In this study, 91 strains of P. ostreatus, including 57 cultivated and 34 wild strains, were analysed using 21 simple sequence repeat markers developed from the genomic sequence of “P. cf. floridanus”. Among the cultivated strains, 46 were found to possess different allelic patterns. The remaining 11 strains were clustered into five groups, each with their own private alleles, suggesting that 10.5% (6/57) of the cultivated strains were previously labelled with improper names. Our analyses indicate that wild strains harbored greater genetic diversity than the cultivated strains. With regard to the cultivation history of P. ostreatus, the cultivated strains in China have three sources: direct introduction from Europe, domestication from wild strains from China, or hybridisation of the European and Chinese strains. Furthermore, we propose a core collection of P. ostreatus with 34 strains, including 13 wild and 21 cultivated strains. The allele retention proportion of the core collection for the entire collection was 100%.     

Novel microsatellite markers suitable for genetic studies in the white button mushroom <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Co-dominant microsatellite molecular markers were obtained from the Agaricus bisporus cultivated mushroom. Their potential for both the molecular characterisation of commercial strains and the monitoring of the intraspecific genetic variation was demonstrated. The analysis of 673 unique sequences issued from public database and 59 from an enriched A. bisporus genomic library resulted in the development of a total of 33 single sequence repeat or microsatellite (SSR) markers. Their usefulness for genetic analysis was assessed on 28 strains, which include six cultivars representative of traditional lineage, two hybrids and 20 strains originating from wild populations. A. bisporus SSR markers displayed each from two to ten alleles, with an average of 5.6 alleles per locus. The observed heterozygosity ranged from 0 to 0.88. Cluster analysis resulting from SSR fingerprintings was in agreement with published A. bisporus population structure. A combination of only three selected SSR markers was sufficient to discriminate unambiguously 27 out of 28 distinct genotypes. However, the two genetically related hybrids were not distinguishable. Multiplexing was tested, and up to seven loci could be genotyped simultaneously. We are therefore reporting the first development in A. bisporus of a set of microsatellite markers powerful and suitable for genetic analysis.     

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.     

Evaluation of Genetic Diversity and Population Structure Analysis among Germplasm of Agaricus bisporus by SSR Markers

Agaricus bisporus is a popular edible mushroom that is cultivated worldwide. Due to its secondary homothallic nature, cultivated A. bisporus strains have low genetic diversity, and breeding novel strains is challenging. The aim of this study was to investigate the genetic diversity and population structure of globally collected A. bisporus strains using simple sequence repeat (SSR) markers. Agaricus bisporus strains were divided based on genetic distance-based groups and model-based subpopulations. The major allele frequency (MAF), number of genotypes (NG), number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), and polymorphic information content (PIC) were calculated, and genetic distance, population structure, genetic differentiation, and Hardy–Weinberg equilibrium (HWE) were assessed. Strains were divided into two groups by distance-based analysis and into three subpopulations by model-based analysis. Strains in subpopulations POP A and POP B were included in Group I, and strains in subpopulation POP C were included in Group II. Genetic differentiation between strains was 99%. Marker AB-gSSR-1057 in Group II and subpopulation POP C was confirmed to be in HWE. These results will enhance A. bisporus breeding programs and support the protection of genetic resources.