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Basal stem rot (BSR) disease is a devastating threat to Malaysia oil palm plantation, which is caused by Ganoderma boninense fungi. Dikaryon is the dominant stage of G. boninense and the pathogenic form of the fungus as compared to the monokaryon. Mating signalling response lead by mitogen-activated pathway (MAPK) responsible in the transformation of sterile monokaryon to pathogenic dikaryon. Expression of the STE3 gene involved in the autocrine activation of mating pathway. Mutations of STE3 gene in some pathogenic yeast resulted in inhibition of pheromone responses and blocked the signalling in the cell fusion. Identification of pheromone receptor, STE3 gene involved in mating signalling pathway is important to understand the mating response towards pathogenesis of G. boninense. Genome walking and polymerase chain reaction (PCR) were carried out to amplify isolate the full length of STE3 gene. Several in silico sequence analysis were conducted to characterise the STE3 gene. The full-length gene sequence including the upstream and downstream regions obtained could be used in future studies to understand pathogenesis of G. boninense.

Plant biomass is one of the main renewable materials on Earth. It is used as substrate by industry to convert it into useful products, such as biofuel and chemicals. Natural forests, forest plantations and agroforestry plantations are recognized as an important reservoir of renewable carbon. Fungi play an important role in plant biomass degradation, also in forest ecosystems. Therefore, improving the production of fungal plant biomass converting enzymes is of great interest to biotechnology. These enzymes have been mainly studied in saprobic ascomycetes, and much less extensively in basidiomycetes. This is largely caused by the better manageability of ascomycetes in industrial fermentations and the availability of transformation systems for many ascomycetes, while these aspects are still a major challenge in basidiomycetes. Despite this, basidiomycetes form a promising source of novel enzymes with putatively different biochemical properties than ascomycete enzymes. Plant biomass consists mainly of polysaccharides, lignin and proteins, forming a complex network of polymers interacting with each other. White-rot basidiomycete fungi are well known for their unique ability to decompose all wood polymers, including the recalcitrant lignin, by secreting hydrolytic and oxidative enzymes. This ability has raised significant interest in white-rot fungi, since lignin removal from plant biomass remains a challenge in industry and academia. In this thesis, I focused on the white-rot fungus Dichomitus squalens as a basidiomycete model for plant biomass degradation, and extend those insights to other white-rot fungi. In the last years, the ability of this species to produce industrially relevant enzymes has been demonstrated, its genome encodes a broad range of enzymes for polysaccharide and lignin degradation, and its ligninolytic potential has been validated in both submerged and solid state cultures. This thesis focused on the genetical and physiological variation among different D. squalens strains in relation to plant biomass utilization and various approaches of the wood degradation process. For this, state of the art molecular and omics technologies were combined with amplified fragment length polymorphisms (AFLP), phylogeny, microscopy and enzyme activities, to demonstrate differences in the plant biomass degradation approach between mono- and dikaryotic strains, as well as between geographically different lineages of the fungus. It also revealed a partial adaptation of D. squalens to different wood types (hard- and softwood). In addition, I elucidated the response of D. squalens to the presence of mono- and oligomers derived from plant biomass and identified two inducers of CAZyme gene expression related to lignocellulose degradation. While these inducers were functional in both mono- and dikaryotic strains, their regulatory patterns showed differences between these strains.

Plant biomass is one of the main renewable materials on Earth. It is used as substrate by industry to convert it into useful products, such as biofuel and chemicals. Natural forests, forest plantations and agroforestry plantations are recognized as an important reservoir of renewable carbon. Fungi play an important role in plant biomass degradation, also in forest ecosystems. Therefore, improving the production of fungal plant biomass converting enzymes is of great interest to biotechnology. These enzymes have been mainly studied in saprobic ascomycetes, and much less extensively in basidiomycetes. This is largely caused by the better manageability of ascomycetes in industrial fermentations and the availability of transformation systems for many ascomycetes, while these aspects are still a major challenge in basidiomycetes. Despite this, basidiomycetes form a promising source of novel enzymes with putatively different biochemical properties than ascomycete enzymes. Plant biomass consists mainly of polysaccharides, lignin and proteins, forming a complex network of polymers interacting with each other. White-rot basidiomycete fungi are well known for their unique ability to decompose all wood polymers, including the recalcitrant lignin, by secreting hydrolytic and oxidative enzymes. This ability has raised significant interest in white-rot fungi, since lignin removal from plant biomass remains a challenge in industry and academia. In this thesis, I focused on the white-rot fungus Dichomitus squalens as a basidiomycete model for plant biomass degradation, and extend those insights to other white-rot fungi. In the last years, the ability of this species to produce industrially relevant enzymes has been demonstrated, its genome encodes a broad range of enzymes for polysaccharide and lignin degradation, and its ligninolytic potential has been validated in both submerged and solid state cultures. This thesis focused on the genetical and physiological variation among different D. squalens strains in relation to plant biomass utilization and various approaches of the wood degradation process. For this, state of the art molecular and omics technologies were combined with amplified fragment length polymorphisms (AFLP), phylogeny, microscopy and enzyme activities, to demonstrate differences in the plant biomass degradation approach between mono- and dikaryotic strains, as well as between geographically different lineages of the fungus. It also revealed a partial adaptation of D. squalens to different wood types (hard- and softwood). In addition, I elucidated the response of D. squalens to the presence of mono- and oligomers derived from plant biomass and identified two inducers of CAZyme gene expression related to lignocellulose degradation. While these inducers were functional in both mono- and dikaryotic strains, their regulatory patterns showed differences between these strains.

Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.

‘Nongjin-go’ is a new breed strain of Lentinula edodes, saw-dust bag variety. It is a cross combination of dikaryon Lentinula edodes ASI 3305mut and monokaryon L5-16 of L. edodes ASI 3305(Sanjo701ho). We crossbred them by 2011 and verified productive capacity from 2012 to 2013 in Rural Development Administration. Optimum temperature of mycelial growth is 30oC and it of fruit-body primodium formation is range from 15 to 23oC. Nongjigo is agricultured at mid-high temperature well. Fruiting body is platy-hemisphere, light brown and centralizing. And bast is formed around edge of pileus. Yield productions per period is regular than ‘Sanjo701’. Plastic bag culture medium is 1.5 kilogram and culture periods are 90~100 days. As its browning of pileus in culture is a little slow, Light and ventilation is needed a lot in light-culturing. Humidity is controlled properly for its color in fruit-body growing. Tested culture medium is consisted of 80% Oak-Tree saw-dust and 20% rice-bran.

‘Baekjung’ adaptable to high temperature was made by crossing between monokaryon derived from selfing of brown strain and monokaryon derived from Korea white strain. In the condition that temperature is maintained at 10oC without low temperature of 4oC suppressing treatment and wrapping during cultivation period, it showed good productivity than Uri1ho(control). The optimum temperature of mycelial growth was 30oC and that of fruiting body initiation and development were 14oC and 7oC, respectively. The days for the fruiting and yield were 7 days and 277±11.2 g per 1,100 ml bottle, respectively. This variety needed high concentration of carbon dioxide up to 4,000 ppm for the good quality.

To develop new variety of oyster mushroom, 63 intra-specific hybrids between the strain Suhan and #Nongi201 were developed using hyphal anastomosis technique in 2004. The Po2008-275 hybrid between the dikaryon strain 04-154(Suhan x #Nongi201) and the monokaryon strain derived from ASI2487 were developed using hyphal anastomosis in 2008. The Po2008- 275 was shown the best cultural characteristics, selected to be a new variety and named as 'Guseol'. The new commercial strain, 'Guseol' had dark grey pilei and grows well under spring and autumn conditions in Korea. The fruiting bodies of 'Guseol' were of an excellent quality in that not only the stipe was thick and long but also the pileus was small and hard. The optimum temperatures for mycelial growth and fruiting body development were 25~30℃ and 10~16℃, respectively. Time period required for the initiation of the first fruiting body was about 3 to 5 days depending on the temperatures. The shape of fruiting body was thin funnel shape. Fruiting body production per box(43x43x12cm) was about 1545±400.9g which was almost 137% quantity compared to that of parental strain 04-154. Relatively low temperature incubation (11℃) resulted in the development of better quality of 'Guseol' mushrooms. When two different media including potato dextrose medium and mushroom complete medium were compared, the mycelial growth of this mushroom were much faster in mushroom complete medium. Similar results were observed with other variety '#Chunchu2'. Analysis of the genetic characteristics of the new commercial strain 'Guseol' showed a major DNA profile as that of the parental 04-154 when primer URP 1, primer URP 2 and primer URP 5 were used, but different to '#Chunchu2' that was used as a control. This new variety of the dark grey oyster mushroom had smart and high quality image that corresponds well to "health food". We therefore expect that this new strain will satisfy the consumers demand for variety and excellent mushrooms.

Frontiers in microbiology 9, 2545 (2018). doi:10.3389/fmicb.2018.02545, Published by Frontiers Media, Lausanne

We performed phylogenetic analysis for ten Pleurotus species, based on internal transcribed spacer (ITS) sequences of rDNA. A phylogenetic tree was constructed on the basis of 31 oyster fungi strains of different origin and 10 reference sequences from GenBank. Our analysis demonstrates that the tested Pleurotus species are of monophyletic origin. We evaluated the evolutionary distances between these species. Classic genetic analysis of sexual compatibility based on monokaryon mon-mon crosses showed no reproductive barriers within the P. cornucopiae-P. euosmus species complex. Thus, despite the divergence (subclustering) between commercial strains and natural isolates of P. ostreatus revealed by phylogenetic analysis, there is no reproductive isolation between these groups. A common allele of the matB locus was identified for the commercial strains Sommer and L/4, supporting the common origin of these strains.

D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.

Auricularia auricula-judae is a traditional edible fungus that is cultivated widely in China. In this study, a genetic linkage map for A. auricula-judae was constructed using a mapping population consisting of 138 monokaryons derived from a hybrid strain (A119-5). The monokaryotic parent strains A14-5 and A18-119 were derived from two cultivated varieties, A14 (Qihei No. 1) and A18 (Qihei No. 2), respectively. In total, 130 simple sequence repeat markers were mapped. These markers were developed using the whole genome sequence of A. auricula-judae and amplified in A14-5, A18- 119, and the mapping population. The map consisted of 11 linkage groups (LGs) spanning 854 cM, with an average interval length of 6.57 cM. A testcross population was derived from crossing between the monokaryon A184-57 (from the wild strain A184 as a tester strain) and the mapping population. Important agronomic trait-related QTLs, including mycelium growth rate on potato dextrose agar for the mapping population, mycelium growth rate on potato dextrose agar and sawdust for the testcross population, growth period (days from inoculation to fruiting body harvesting), and yield for the testcross population, were identified using the composite interval mapping method. Six mycelium growth raterelated QTLs were identified on LG1 and LG4, two growth period-related QTLs were identified on LG2, and three yieldrelated QTLs were identified on LG2 and LG6. The results showed no linkage relationship between mycelium growth rate and growth period. The present study provides a foundation for locating genes for important agronomic characteristics in A. auricula-judae in the future.

Clitopilus passeckerianus is the fungal species responsible for the production of pleuromutilin, a diterpene antibiotic that is gaining in commercial interest. Production of the antibiotic is constrained by the low titers typically obtained from isolates. We therefore set out to investigate the possibility of using classical breeding techniques coupled with genetic manipulation as a means to develop such fungi. We show that the original production strain of C. passeckerianus is able to fruit under laboratory conditions, giving viable haploid meiotic basidiospores. The derived progeny displayed the typical physiological and genetic characteristics of a tetrapolar mating system. The monokaryon haploids produced pleuromutilin and haploid lines were amenable to genetic manipulation. Together this shows that the basic requirements for a classical breeding approach are present and the tools required to undertake directed genetic engineering on haploid strains are available, demonstrating that strain improvement may be feasible in this fungus.

This research was carried out to evaluate whether gamma ray is a useful tool for breeding new strains of mushrooms. For this research, 5 mutant groups, 20 strains of Hypsizigus marmoreus, 2 strains of Lyophyllum decastes, and 1 strain of Lyophyllum shimeji were used. Monokaryon spores from one variety of H. marmoreus were irradiated with 50~2,000 Gy of gamma ray. The propriety dose was 50~200 Gy for mutagenesis. Mutant monokaryon mycelia crossed each order to become dikaryon mycelia. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR, and the products were sequenced. The sequences of the ITS regions (16 partial rDNA, complete ITS1, 5.8 rDNA and partial rDNA) were analyzed by PCR, and strains of H. marmoreus, L. decastes, and L. shimeji were auto- sequenced. The lengths of the sequenced ITSs were 1,052~1,143 nucleotides. Genetic matrices were cal-culated using Nei-Li's genetic distance coefficient based on ITS sequence. The dissimilarities were 0~3.35% in strains of H. Hypsizigus. In addition, a phylogenetic tree was constructed based on ITS se-quences using the neighbor-joining (NJ) method. The phylogenetic tree revealed that 23 strains and 5 mutant groups were divided into 12 clusters; the mutant groups fell into different clusters. These results show that mushroom spores were mutated effectively by gamma ray; therefore, gamma ray could be a useful tool for breeding new strains of mushrooms.

The primary objective of the present study is the characterization of the somatic hybrids of dikaryon-monokaryon (dimono) crosses in mushroom breeding. We employed this technique for developing superior strains from Pleurotus ostreatus strains with 48 intraspecific hybrids of 12 combinations between six P. ostreatus strains and one P. florida strain. The results on the experiments of hybridization rate, nuclear DNA patterns, and colors and morphology of fruit-bodies, are presented. In di-mono crosses, somatic hybrids among Pleurotus strains showed 100% of crossability as seen in those between P. ostreatus and P. florida strains indicating that the nuclei of a dikaryon is inferred to be migrated to a recipient. 87.5% of the somatic hybrids among Pleurotus strains were similar to the donor dikaryons, and 12.5% of the somatic hybrids presented DNA patterns of both parents. In 16.6% of di-mono crosses between P. ostreatus and P. florida, the nuclear DNA patterns of all hybrids showed the same or similar patterns compared to the donor dikaryons. 70.9% of the hybrids between P. ostreatus and P. ostreatus were similar to the donor dikaryons and 12.5% of them presented the DNA patterns of both parents. 79.2% of fruiting body morphology of the hybrids among Pleurotus strains were similar to the dikaryons and 20.8% of them were similar to both parents. Interestingly, the morphology of all dikaryons were dissimilar each other. All hybrid strains between dikaryon P. florida and monokaryon P. ostreatus showed the fruiting body of which colors were similar to those of the dikaryon, while the hybrids between dikaryon P. ostreatus and monokaryon P. florida were showed the combined colors of both parents. Therefore, the fruiting body color of P. florida tends to be generally dominant. In conclusion, the present study provides a way to find out and suggest superior hybrid strains using the nuclear DNA patterns of hybrids between Pleurotus strains as well as the characteristics of their fruiting bodies. The advantages of the di-mono crossing are needs to be fully utilized in mushroom breeding because it is an ideal way to develop the superior strains of Pleurotus.

The morphological variation of basidiomata of Filoboletus manipularis (Berk) Singer collected in southern Viet Nam was studied. Phylogenetic analyses comprising three gene loci indicated that these collections, although exhibiting widely varying morphologies, represented a single species with a population composed of genetically diverse, sexually compatible monokaryon parental strains. No correlation was found between any aspect of morphological variation and intraspecific phylogenetic patterns for the three gene loci studied. Primers were designed to amplify the intron-rich 5′ region of the translation elongation factor 1-α gene (tef1α) and amplicons cloned and sequenced to characterize the parental haplotypes for individual basidiomata. The presence of recombination over the entire morphological diversity seen was confirmed by split decomposition analysis and analysis of gene diversity indicated a lack of allelic fixation within local populations. On several occasions, more than two apparent parental haplotypes were characterized from individual basidiomata, indicating that at least some basidiomata are chimeric or otherwise develop from a multinucleate condition. The literature supporting our observations of the occurrence of multinucleate basidiomata is reviewed and possible mechanisms for this phenomenon are proposed.

Phylogenies based on ITS and LSU sequences show that the Sparassis crispa complex comprises several monophyletic clades, in some cases co\rresponding to named taxa (i.e. S. crispa, S. radicata), but others lacking names (i.e. eastern and southwestern North American S. “crispa”). In our study, morphological examination of numerous collections also distinguished subtle differences correlated with geographic distribution. Underlying these problems, several taxa lacked type specimens for taxonomic analysis. In this paper, appropriate epitypes are designated and names assigned. Extensive sexual compatibility experiments, described within, indicate that monokaryon, haploid isolates of collections from North America and Europe are consistently sexually compatible to some degree. Inherent in the study, different “species concepts” were tested, with the “biological species concept,” based on sexual compatibility, being the least restrictive. We propose two new taxa, S. americana and S. americana f. arizonica.

3 p.-2 tab. Gaskell, Jill et al., We report the genome of Postia (Rhodonia) placenta MAD-SB12, a homokaryotic wood decay fungus (Basidiomycota, Polyporales). Intensively studied as a representative brown rot decayer, the gene complement is consistent with the rapid depolymerization of cellulose but not lignin., The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. BH was funded by the AMIDEX foundation (MicrobioE project, grant number ANR-11-IDEX-0001-02).

International audience; We report the genome of Postia (Rhodonia) placenta MAD-SB12, a homokaryotic wood decay fungus (Basidiomycota, Polyporales). Intensively studied as a representative brown rot decayer, the gene complement is consistent with the rapid depolymerization of cellulose but not lignin.

Pleurotus eryngii (DC.) Quel. is widely used for bioconverting lignocellulosic byproducts into biofuel and value added products. Sequencing and annotating the genome of a monokaryon strain P. eryngii 183 allows us to gain a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases for degradation of lignocellulose in white-rot fungi. The genomic data provides insights into genomic basis of degradation mechanisms of lignin and cellulose and may pave new avenues for lignocellulose bioconversion.

Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

Flammulina velutipes is a potentially excellent fungus to study basic mechanisms of basidiomycete mycelium biology. To provide a better understanding of the mechanism of hyphae growth and fruit-body formation, the biological functions of the differentially abundant proteins between the fruiting dikaryon and the non-fruiting monokaryon of F. velutipes were investigated at the proteomic level using iTRAQ-coupled two-dimensional liquid chromatography tandem mass spectrometry technique. Among the 1198 proteins identified with high confidence, a total of 472 proteins were detected differentially abundant at least one of the mycelium development stages. In-depth data analysis revealed that differentially expressed proteins were influenced a variety of cellular processes, particularly metabolic processes. Functional pathway analysis indicated that 63 up-regulated proteins at only the fruiting dikaryon (Fv13) stage were mainly distributed in 51 specific Kyoto Encyclopedia of Genes and Genome pathways, such as amino acids biosynthesis and metabolism, signaling pathway, and central carbon metabolism. These up-regulated proteins could possibly serve as potential biomarkers to study the mycelium development pathways as well as provide new insights on the mycelium heterogenic compatibility and fruit-body formation mechanisms of basidiomycetes.

This study was carried out to investigate the morphological and physiological characteristics of six new cultivars of Hypsizygus marmoreus (Hm) and measure endo-, exo-cellular enzyme-specific activity. The domestic wild stain (Hm3-10) and commercial strain in Japan (Hm1-1) were mated by crossing monokaryon mycelia. We gained 58 strains from one of 400 crosses through the cultivation experiment, and selected six strains from one of 58 strains through the cultivation experiment. When six of the selected new strains were grown during several spawn culture periods (60, 70, 80, 90, and 100 days), a spawn culture period of more 80 days was considered to be excellent as being shorter than 19~20 days. Therefore, we determined the period of spawn culture as 80 days. Three strains such as Hm15-3, Hm15-4, and Hm17-5 showed an excellent result. When endo-cellular enzyme activity measured eight strains, we obtained a result of that specific activity of -amylase at the highest as 73.9~102.2 unit/mg protein, and chitinase is lower than -amylase at 8.1~13.1 unit/mg protein. When exo-cellular enzyme activity measured eight strains, we determined the result of that specific activity of -amylase is the highest at 5,292~1,184 unit/mg protein, and CMCase and xylanase were 1,140~245 unit/mg protein, 94~575 unit/mg protein, compared to each other. However, the enzyme activity of -glucosidase and chitinase is low.

Pathogenicity tests were carried out on oil palm seedlings and germinated seeds using monokaryotic and dikaryotic mycelia in order to investigate the indirect role of basidiospores in initiating basal stem rot disease in oil palm. The pathogenicity tests conducted showed that monokaryotic mycelia were not able to cause infection while reconstituted dikaryotic mycelia could initiate basal stem rot infection on both oil palm seedlings and germinated seeds. The severity of the disease symptoms on both seedlings and germinated seeds was found to be related to the size of the inoculum, with larger inoculum resulting in more severe symptoms. The present study indicates that monokaryotic mycelia which originated from germinating basidiospores could colonise any available substrate and that basidiospores therefore indirectly played a role in basal stem rot infection in oil palm fields.

We expect that sexual selection may play an important role in the evolution of mushroom-forming basidiomycete fungi. Although these fungi do not have separate sexes , they do play female and male roles : the acceptance and the donation of a nucleus, respectively. The primary mycelium (monokaryon) of basidiomycete fungi, growing from a germinating sexual spore, is hermaphroditic, but it loses female function upon the acceptance of a second nucleus. The resulting dikaryon with two different nuclei in each cell retains a male potential as both nuclei can fertilize receptive mycelia. We tested the occurrence of sexual selection in the model species of mushroom-forming basidiomycetes, Schizophyllum commune , by pairing monokaryons with fully compatible dikaryons. In most pairings, we found a strong bias for one of the two nuclei although both were compatible with the monokaryon when paired alone. This shows that sexual selection can occur in mushroom-forming basidiomycetes. Since the winning nucleus of a dikaryon occasionally varied depending on the receiving monokaryon, we infer that sexual selection can operate through choosiness of the receiving individual (analogous to female choice). However, in other cases the same nucleus won, irrespective of the receiving monokaryon, suggesting that competition between the two nuclei of the donating mycelium (analogous to male–male competition) might also play a role.

Pleurotus spp. is one of the most important cultivated mushrooms in the world by their nutrition and medicinal property. In Vietnam, although many Pleurotus species are popularly cultivated, but most of them are imported. These strains are easily degenerated when they are cultivated in large scale, because they are difficult to adapt to the environment of Vietnam. This research aims to construct the monokaryon collection of Pleurotus spp. with details of taxonomy, genetic diversity, cultivating traits, monokaryotic mating types focused on Pleurotus strains with the high yield, genetic stable and adaptability to Vietnam environment. 09 Pleurotus strains were collected in South Vietnam and signed PL1 to PL9. Identification by morphology and phylogenetic analysis based on ITS sequences showed that PL1 is P. citrinopileatus; PL2, PL6 and PL9 are P. ostreatus; PL3 is P. cystidiosus; PL4 and PL8 are P. pulmonarius; PL5 is P. cornucopiae; PL7 is P. incarnatus. AFLP analyses indicated a wide genetic diversity of the collected strains with the similar coefficient 52 – 90 %. Pair of PL2 and PL9 is the most closed genetic distance and pair of PL6 and PL7 is the furthest genetic distance. PL4, PL8 and PL1 adapted well to Vietnam environment. PL2, PL5, PL6 and PL9 are cold strains and formed fruiting bodies slowly. PL3 had the highest biological efficiency, but the longest harvesting period (2.5 – 3 months). Except PL7, the biological efficiencies of other strains are over 50 %. 120 monokaryotic isolates of PL1, PL2 and PL8 are collected and determined the mating type.

Temporal and spatial variations in airborne spore concentrations of selected allergenic and pathogenic fungi were examined in Dublin, Ireland, in 2005. Air samples were taken at four outdoor locations in the city every 2 weeks, coupled with measurements of meteorological conditions. Total culturable airborne fungal spore concentrations in Dublin ranged from 30–6800 colony forming units per cubic metre of air (CFU m −3 ) over the 12-month period. Cladosporium , Penicillium , Aspergillus and Alternaria spores were constantly present in the Dublin atmosphere, representing >20% of the total culturable spore count. Concentrations of Cladosporium increased significantly in summer and reached allergenic threshold levels, peaking at over 3200 CFU m −3 in August. Penicillium spore concentrations never reached allergenic threshold levels, with average concentrations of −3 . Alternaria conidia formed only 0.3% of the total culturable fungal spore count and concentrations never exceeded 50 CFU m −3 , attributable to the coastal position of Dublin and its low levels of arable production. The opportunistic human pathogen Aspergillus fumigatus was present throughout the year in nominal concentrations ( −3 ), but sporadic high counts were also recorded (300–400 CFU m −3 ), the potential health implications of which give cause for concern. Spores of neither Cryptococcus neoformans nor Stachybotrys chartarum were detected, but airborne basidiospores of Schizophyllum commune were evidenced by the dikaryotization of monokaryon tester strains following exposure to the air. The relationships between airborne fungal spore concentrations and meteorological factors were analysed by redundancy analysis and revealed positive correlations between temperature and Cladosporium and relative humidity and Penicillium and Aspergillus .

Production of mitotic spores has been reported for various species of the Agaricomycetes but is generally rather neglected and the extent of asexual sporulation in this class of Basidiomycetes is unknown. The typical life cycle of Agaricomycetes comprises of two alternate mycelial stages, the sterile monokaryon and the fertile dikaryon. The monokaryon contains only one type of haploid nuclei. The dikaryon is formed by mating of two compatible monokaryons and has two distinct haploid nuclei in its hyphal cells, each one per parental monokaryon. Both types of mycelia may produce asexual spores but species differ in whether mitospores are formed at the monokaryon, at the dikaryon, at both or at none. Mitospores produced on the dikaryon might be homokaryotic or heterokaryotic. Here, we present an overview on species of the Agaricomycetes and their spores. Types of sporulation include thallic spore formation (arthroconidia), blastic sporulation (blastoconidia) and intracellular production of thick-walled chlamydospores. Where known, we discuss functions of spores and regulation of mitospore production.

The original publication is available at www.springerllink.com., Article, Journal of Wood Science. 53(4):339-343 (2007)

Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13 × 3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13 × 3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.

Heterothallic mushrooms accomplish sex by exchanging nuclei without cytoplasm. Hyphal fusions occur between haploid mycelia resulting from germinated spores and subsequent reciprocal nuclear exchange without cytoplasmic mixing. The resulting dikaryon is therefore a cytoplasmic mosaic with uniformly distributed nuclei (two in each cell). Cytoplasmic inheritance is doubly uniparental: both mated monokaryons can potentially transmit their cytoplasm to the sexual spores, but normally only a single type per spore is found. Intracellular competition between mitochondria is thus limited, but at the dikaryon level, the two types of mitochondria compete over transmission. This creates the conditions for genomic conflict: within the dikaryon, a selfish mitochondrial mutant with increased relative transmission can be favoured, but selection between dikaryons will act against such a mitochondrial mutant. Moreover, because nuclear fitness is directly dependent on dikaryon fitness, a reduction in dikaryon fitness directly conflicts with nuclear interests. We propose that genomic conflict explains the frequent occurrence of non-reciprocal nuclear exchange in mushrooms. With non-reciprocal exchange, one monokaryon donates a nucleus and the other accepts it, but not vice versa as in the typical life cycle. We propose a model where non-reciprocal nuclear exchange is primarily driven by mitochondria inducing male sterility and the evolution of nuclear suppressors.

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.

The mating system of Stropharia rugoso-annulata Farlow apud Murrill was studied by pairing single spore isolates from the same fruitbody, and the genetic diversity of monokaryotic strains with different mating types was evaluated by the RAPD technique. Basidiospores could germinate normally on PGP (potato/glucose/peptone) medium at 30 °C. Analysis of self- and cross-pairings revealed that Stropharia rugoso-annulata was heterothallic and tetrapolar. RAPD analysis detected polymorphism among monokaryotic strains, with more genetic variation within monokaryotic strains with non-parental mating type compared to monokaryotic strains with parental mating type. These results were in general agreement with the existing knowledge, confirming the validity and usefulness of the RAPD technique. Therefore, the RAPD technique will provide an exciting and valuable tool for a large-scale study on identification and genetic resources of monokaryotic strains, and should lead to a more efficient understanding and utilization of genetic diversity of monokaryotic strains in cross breeding by breeders.

The heterotrimeric G-protein α-subunit family plays multiple roles in eukaryotic cells, such as the regulation of growth and development, of pathogenicity and of the transmission of pheromone stimulation. In the homobasidiomycete Schizophyllum commune, some genes encoding heterotrimeric G-protein α-subunits (SCGPα1, SCGPα2, ScGP-A, ScGP-B and ScGP-C) have been reported. In this study, constitutively active mutants of ScGP-A, ScGP-B and ScGP-C were generated by site-directed mutagenesis and introduced into the S. commune monokaryon strain to investigate the function of each gene. Northern analysis showed that the mutated genes were strongly expressed when compared with endogenous G-proteins in many clones. Upon macroscopic examination, some transformed clones expressing ScGP-A (Q207R) and ScGP-C (Q204R) mutant genes exhibited a slight suppression of aerial-hyphae formation in the monokaryon strain. In contrast to the slight suppression of aerial-hyphae formation in the monokaryon, most clones expressing mutated ScGP-A or ScGP-C genes failed to form fruit-bodies in the dikaryon strain. This observation indicated that ScGP-A and ScGP-C played a role in suppressing fruit-body formation in the dikaryon. Furthermore, these phenotypes were similar to the phenotype of the thn mutant in S. commune to some extent. Since the thn-1 gene encodes a putative regulator of the G-protein signalling protein (RGS), ScGP-A and ScGP-C might be targets of thn-1.

Flammulina velutipes is one of the major edible mushrooms in the world. Recently, abnormalities that have a negative impact on crop production have been reported in this mushroom. These symptoms include slow vegetative growth, a compact mycelial mat, and few or even no fruiting bodies. The morphologies and fruiting capabilities of monokaryons of wild-type and degenerate strains that arose through arthrospore formation were investigated through test crossing. Only one monokaryotic group of the degenerate strains and its hybrid strains showed abnormal phenotypes. Because the monokaryotic arthrospore has the same nucleus as the parent strain, these results indicated that only one aberrant nucleus of the two nuclei in the degenerate strain was responsible for the degeneracy. A sequence-characterized amplified region marker that is linked to the degenerate monokaryon was identified based on a polymorphic sequence that was generated using random primers. Comparative analyses revealed the presence of a degenerate-specific genomic region in a telomere, which arose via the transfer of a genomic fragment harboring a putative helicase gene. Our findings have narrowed down the potential molecular targets responsible for this phenotype for future studies and have provided a marker for the detection of degenerate strains.

Several hundred monokaryotic and new dikaryotic strains derived thereof were established from (+)-valencene tolerant Pleurotus species. When grouped according to their growth rate on agar plates and compared to the parental of Pleurotus sapidus 69, the slowly growing monokaryons converted (+)-valencene more efficiently to the grapefruit flavour compound (+)-nootkatone. The fast growing monokaryons and the slow × slow and the fast × fast dikaryotic crosses showed similar or inferior yields. Some slow × fast dikaryons, however, exceeded the biotransformation capability of the parental dikaryon significantly. The activity of the responsible enzyme, lipoxygenase, showed a weak correlation with the yields of (+)-nootkatone indicating that the determination of enzyme activity using the primary substrate linoleic acid may be misleading in predicting the biotransformation efficiency. This exploratory study indicated that a classical genetics approach resulted in altered and partly improved terpene transformation capability (plus 60%) and lipoxygenase activity of the strains. Fil: Omarini, Alejandra Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Leibniz Universität Hannover. Hannover; Alemania Fil: Plagemann, Ina. Leibniz Universität Hannover. Hannover; Alemania Fil: Schimanski, Silke. Leibniz Universität Hannover. Hannover; Alemania Fil: Krings, Ulrich. Leibniz Universität Hannover. Hannover; Alemania Fil: Berger, Ralf G.. Leibniz Universität Hannover. Hannover; Alemania

Cryptococcus neoformans and Cryptococcus gattii are closely related basidiomycetous fungi that commonly infect humans to predominantly cause meningoencephalitis. Both grow as budding yeasts in the environment and in the infected host yet undergo a dimorphic transition to a filamentous monokaryon or dikaryon during sexual reproduction. This chapter covers recent exciting advances in the field with special consideration of features of the virulence and life cycle relevant to studies of filamentous fungi and the emergence of microbial pathogens successfully infecting animals. The iron regulatory network and iron acquisition functions have been examined in some detail for C. neoformans. Initially, the response of the fungus to iron deprivation was examined by transcriptional profiling, and this study identified general patterns of gene expression as well as specific iron-responsive functions. The latter genes encoded iron acquisition functions and a predicted mannoprotein described as a cytokine-inducing glycoprotein (Cig1). The availability of the genome sequences and molecular techniques for C. neoformans strains provides an opportunity to define the transcriptome of the pathogen under a variety of growth conditions. An interesting recent study on extracellular proteome targeted proteins associated with extracellular vesicles. The contribution of the α allele to pathogenicity is background dependent, and virulence is a quantitative trait, in which mating-type locus (MAT) interacts with other unlinked genes to contribute to virulence.

Historically, Coprinopsis cinerea (C. lagopus sensu Buller, C. macrorhizus) and Schizophyllum commune were the first hymenomycete species shown to be heterothallic, and it is not surprising that studies on mating type have, until recently, focused on these two species. It was Hans Kniep who recognized that the matingtype genes were multiallelic and many different mating types existed in the population. Kniep’s remarkably thorough studies, involving analyzing progeny from many generations of crosses, led him to note that some 1 to 2% of the progeny of sexual crosses in S. commune had new mating types, and he naturally attributed this to mutation. Haploid basidiospores germinate to give a self-sterile mycelium that is generally called a monokaryon because it has predominantly uninucleate cells. Mushrooms appear to be unique among fungi in having evolved multiple versions of their pheromones and receptors, and this has been driven by the need to increase the numbers of mating types. The genome sequence of C. cinerea reveals several clusters of genes of related function, suggesting that gene amplification has contributed to many aspects of the biology of this fungus. The majority of hymenomycetes are heterothallic, and it seems likely that as in ascomycetes, homothallic species were derived from heterothallic species. With the molecular and genomic tools we now have, exploring deeper into the study of the MAT loci enhances one's understanding of the importance of sex and its involvement in genome evolution.

Patterns of protein expression in the monokaryons and the derived dikaryon of the basidiomycete Flammulina velutipes were investigated using two-dimensional polyacrylamide gel electrophoresis and silver staining. Over 300 spots were detected on each gel, and 19 proteins were expressed in the dikaryon before fruiting treatment but not expressed in the monokaryon before fruiting treatment. On the other hand, 9 proteins were expressed in the monokaryon specifically. Newly expressed proteins increased after fruiting treatment (at 7 d and 14 d), but decreased after fruit bodies were formed (at 21 d). The number of newly expressed proteins after the fruiting treatment was similar in the monokaryon. Proteins expressed characteristically were classified into 7 types. The highest number of proteins (13 proteins) were specifically expressed in the dikaryon at 14 d when the fruit-bodies were formed. Twenty-seven proteins were expressed specifically in the fruit body. Fruit body specific proteins Pf1 and Pf3 were sequenced. This revealed Pf1 and Pf3 had amino acid sequences that were similar to each other, as well as a high similarity to the deduced amino acid sequence of the FDS gene isolated from F. velutipes.

The karyotype ofFlammulina velutipes (Curt.: Fr.) Sing. was analyzed electrophoretically using contourclamped homogeneous electric field gel electrophoresis and hybridization with DNA probes. The chromosomal DNA from the monokaryon (Fv-4K) and the dikaryon (Fv-4) were resolved into six and eight bands, respectively. The sizes of the chromosomes ranged from 1.9 to 6.0 megabase (Mb) pairs. Each of the separated bands of chromosomal DNA was identified by use of five cloned probes. The number of these chromosomes was estimated to be 6 and 12, respectively; and the size of the entire genome was estimated to be about 20.1 and 38.6Mb, respectively. From a comparison of the hybridization patterns, the existence of allelic chromosomes of different sizes was deduced in the Fv-4 strain.

A suitable screening method for heterozygous DNA markers in shiitake,Lentinula edodes (Berk.) Pegler, is reported. Monokaryons were derived from a dikaryon by de-dikaryotization via protoplast formation. Compatibility of the monokaryons was determined by pairwise culture on agar plates. We selected the primers to amplify polymorphic fragments among the original strain (Hokken600∶H600) and two monokaryons (H600PP-39 and H600PP-67) showing compatibility. A total of 135 fragments were selected as specific random amplified polymorphic DNAs (RAPDs) resulting from 56 primers of the 147 primers tested. Furthermore, we tested whether the polymorphic fragments segregated into 2∶2 among four strains isolated from a basidium. Most of the polymorphic fragments (about 97.8%) showed 2∶2 segregation among the four strains. We concluded that the polymorphic fragments were heterozygous if they were detected in either of the monokaryons (H600PP-39 and H600PP-67) and segregated to 2∶2 among four meiotic strains (H600B-1,-2, -3, and -4). A total of 132 heterozygous DNA markers were therefore selected from a dikaryon of shiitake (Hokken600∶H600).

Using arbitrary primers for a PCR reaction on cDNA from different mating interactions, genes differentially expressed in a mating-type dependent manner have been identified. The cDNAs were prepared from mRNA extracted from liquid grown cultures of the fungus in order to prevent further steps in development and increase the possibility of identifying genes directly under control of the mating type system. A reaction using a monokaryotic strain was compared to a semicompatible mating reaction induced for B-regulated development and a reaction using a fully compatible mating. In the comparison of silver-stained, non-denaturing polyacrylamide gel separation of the PCR products bands differentially detected between the samples were cloned and subjected to further analysis. Bands were characterized which were present in both mating reactions but absent in the monokaryon while the third band was specifically absent in the fully compatible mating interaction. Sequence analysis allowed design of specific primers for verification of differential expression in a semi-quantitative PCR. Thus, use of radioactivity was not necessary for the fast and reproducible method identifying genes specifically regulated by mating reactions.

Recently published taxonomic circumscriptions for taxa withinFlammulina encouraged mating studies to confirm or reject these names or taxa. Three categories of pairing experiments were performed: 1) self-crosses of monokaryon isolates of examplars of various putative taxa; 2) inter-exemplar pairings among exemplar strains; and 3) pairings between examplars and 87 unidentified strains which were grouped on this basis. Mating experiments could distin-guishF. velutipes from other taxa, but not among infraspecific taxa ofF. velutipes (vars.velutipes, lactea, andlupinicola) and monokaryons of all these taxa were partially compatible with those ofF. ononidis. Likewise, isolates ofF. rossica andF. elastica were partially compatible with one another but incompatible with those of other taxa. All other taxa (F. mexicana, F. stratosa, F. populicola, F. fennae) appeared to be genetically isolated. Low levels of interspecific hybridization betweenF. velutipes andF. populicola, and betweenF. velutipes andF. rossica/elastica were also noted.

Four sib-monokaryons and two reconstituted dikaryons of two basidiomycetes,Lenzites betulinus andTrametes hiruta, accelerated the seed germination ofErythrorchis ochobiensis, an achlorophyllous orchid. All isolates ofL. betulinus and three isolates ofT. hirsuta induced the development of plants from germinated seeds. Although three monokaryotic isolates ofT. hirsuta failed to induce the development of plants, the reconstituted dikaryons induced the development.

Laccase and other ligninolytic enzyme higher-producing dikaryons of Pleurotus ostreatus were obtained after crossing of compatible basidiospore-derived monokaryons selected from the parental basidiospore population on the basis of exceptionality in enzyme production, mycelium extension rate and/or colony morphology. As all detected changes in enzyme activity, mycelium extension rate, colony appearance and degradation of the polymeric dye Poly B411 were relatively stable after repeated testing, the dikaryotic isolates prepared in this way seem to be useful for the future biotechnological exploitation. No correlation between the colony appearance or the mating type and the enzyme activity or other characteristics tested has been found.