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Primordia formation is the first and most critical step in the development of fruiting bodies of edible fungi. In this study, the effects of exogenous ascorbic acid (ASA) on the Pleurotus ostreatus mycelia growth and primordia formation were researched and the results showed that the growth rate of P. ostreatus mycelia was accelerated and the time of primordia formation was advanced. The protein content and ascorbate oxidase (AAO) activity analysis showed that with the increase of ASA concentration, the protein content of mycelia first decreased and then increased, and in a certain concentration range, exogenous ASA could significantly promote the activity of AAO. Further expression analysis of the development regulating genes (Pofst3 and Pofst4) as well as blue light receptor coding genes (PoWC-1 and PoWC-2) showed the expression levels of those four genes all changed after the exogenous ASA addition, which indicated that the expression changes of PoWC-1 and PoWC-2, two key genes in the light morphogenesis, might affect the expression levels of development regulating genes Pofst3 and Pofst4, so as to lead to the formation of primordia in advance.

Only a few transcription factors involved in mushroom development have been reported till now, Therefore, identification of transcription factors in common edible mushroom has commercial and scientific importance. In this study, the Pofst3 gene from Pleurotus ostreatus was cloned and characterized. Bioinformatics analysis showed that Pofst3 protein had 71% sequence similarity with fst3 of model mushroom Schizophyllum commune. Furthermore, the function of Pofst3 gene was analyzed by overexpression and antisense silencing in P. ostreatus via Agrobacterium-mediated transformation. Expression verification of Pofst3 in transformants through qRT-PCR showed that compared with the wild type strains, the transcription level was about 1.26 ∼ 9.59 and 0.01 ∼ 0.30 fold in Pofst3 overexpressing and silencing strains, respectively. Petri dish and bag cultivation tests of transgenic strains showed that the number of primordia and the type of fruiting bodies of Pofst3 overexpressing strains were consistent with the wild type strains, i.e. fewer primordia and larger fruiting bodies; the number of primordia formed by Pofst3 silencing strains were more than those of wild type strains, but fruiting bodies were smaller. It was very likely that Pofst3 was involved in the regulation of P. ostreatus development through inhibiting the formation of clusters of primordia.

1-aminocyclopropane-1-carboxylic acid (ACC) is a strong metabolism-dependent chemoattractant for the plant beneficial rhizobacterium Pseudomonas sp. UW4. It is unknown whether enhancing the metabolic rate of ACC can intensify the chemotaxis activity towards ACC and rhizocompetence. In this study, we selected four promoters to transcribe the UW4 ACC deaminase (AcdS) gene in the UW4 &Delta, AcdS mutant. PA is the UW4 AcdS gene promoter, PB20, PB10 and PB1 are synthetic promoters. The order of the AcdS gene expression level and AcdS activity of the four strains harboring the promoters were PB20 &gt, PA &gt, PB10 &gt, PB1. Interestingly, the AcdS activity of the four strains and their parent strain UW4 was significantly positively correlated with their chemotactic activity towards ACC, rhizosphere colonization, roots elongation and dry weight promotion. The results released that enhancing the AcdS activity of PGPRenable them to achieve strong chemotactic responses to ACC, rhizocompetence and plant growth promotion.

Blue light is necessary for initiation of mushroom formation of Pleurotus ostreatus. In this study, we isolated homologues of the blue light receptor genes wc-1 and wc-2 from P. ostreatus, PoWC-1 and PoWC-2. The PoWC-1 contained three typical PAS domains and one PAS domain exhibited significant similarity to the LOV domain of known blue light receptors. The PoWC-2 had one typical PAS domain and one ZnF domain. The qRT-PCR analysis showed that PoWC-1 and PoWC-2 expression increased in a short time, and the final level tended to be stable along with the light illumination. The PoWC-1 and PoWC-2 expression levels of the primordium period was higher than that of mature fruiting-body period; and in the pileus were the highest, followed by the stipe and the gills. The expression of PoWC-1 and PoWC-2 in pre-primordial mycelia was induced by light exposure. In vivo analysis through yeast two-hybrid experiment disclosed that PoWC-1 and PoWC-2 could form heterologous complex to activate the reporter genes and the complex perform the transcription factor function requiring the addition of FAD.

ADDITIONAL INDEX WORDS. enzyme, respiration intensity, cultivation, Pleurotus ostreatus SUMMARY. We investigated a practical method for immobilizing liquid spawn of oyster mushroom (Pleurotus ostreatus) to prolong the storage time and provide convenient transportation of liquid spawn of edible mushrooms. The method was based on the mycelial pellets of liquid spawn adsorbed in carriers. Selected carriers were similar to cultivation substrates, and the best carrier was a mixture of cottonseed hull, corn core, and wheat bran with a ratio of 4.5:4.5:1 by weight. Immobilized spawn were prepared by mixing the pellets from liquid spawn with carriers using a ratio of 1:8 by weight. Within the first 15 days of storage at 20‐25 � C, the immobilized spawn grew strongly, respiration intensity and cellulase activitiesroserapidly,andthecountandbrightnessoftheisozymebandsofesterase, peroxidase, and polyphenol oxidase increased remarkably as well. From days 30 to 60, the cellulase activities fell and the brightness of the peroxidase and polyphenol oxidase bands gradually decreased, whereas the respiration intensity and the band count of esterase and peroxidase remained constant. After 60 days, the cultivated characteristics of the immobilized spawn were same as the fresh conventional solid cottonseed hull spawn. The results showed that immobilized spawn on the basis of the mycelial pellets of liquid spawn adsorbed in carrier can be used to extend the storage time and simplify transportation of liquid spawn of edible mushroom.

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.

Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.

Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster mushroom, Pleurotus ostreatus, plasmid pSHC, pAN7-1, and pBHt1 were evaluated through PEG/CaCl(2)-mediated protoplast transformation. Plasmid pSHC is a newly constructed hph gene expression system, composed of Escherichia coli hph gene, the P. ostreatus sdi promoter, and the CaMV35S terminator. The vector pAN7-1 was commonly used for integrative transformation in filamentous fungi. Plasmid pBHtl is a T-DNA binary vector, usually introduced into fungi by Agrobacterium-mediated transformation. The results showed that plasmids pSHC, pAN7-1, and pBHt1 were all integrated into the host chromosomes and expressed hygromycin B resistance in P. ostreatus. pAN7-1 had the highest transformation efficiency and hph gene expression level, pSHC the second, and pBHt1 the lowest. Growth rates of the transformants on plates containing hygromycin B were in correspondence with their hph gene expression levels. To our knowledge, this is the first report on integrated transformation of plasmid pAN7-1 and pBHt1 in P. ostreatus.

Many cultivated mushroom strains, such as Pleurotus ostreatus TD300, displayed symptoms of degeneration. A spherical virus POSV and four dsRNA segments were extracted from mycelium of P. ostreatus TD300. POSV had a diameter of 23 nm and encapsidated a 2.5 kb dsRNA segment with coat proteins whose molecular weights were 39 kDa and 30 kDa. Four dsRNA segments were 8.2 kb, 2.5 kb, 2.0 kb, and 1.1 kb in size, respectively. The 1.1 kb dsRNA segment often escaped detection. The cDNA and the amino acid sequences of the 8.2 kb dsRNA were homologous to those of RNA-dependent RNA polymerases (RDRP) of ssRNA oyster mushroom spherical virus (OMSV), and contained conserved motifs A to D which were almost identical to those in RDRP of OMSV. The cDNA and amino acid sequences of the 2.5 kb and 2.0 kb dsRNA segments were homologous to that of RDRP and capsid protein of dsRNA virus P. ostreatus virus 1 (PoV1), respectively. In particular, the amino acid sequence of 2.5 kb dsRNA segment had high identity with the conserved motifs A to C in RDRP of PoV1, a Partiviridae virus. After eliminating the viruses in P. ostreatus TD300, the symptoms of degeneration completely disappeared. The results reveal that P. ostreatus TD300 was at least infected by a particle virus POSV, and two naked viruses, one was a dsRNA virus with a 2.0 kb dsRNA segment, the other was an ssRNA virus whose replicating form of genome was an 8.2 kb dsRNA segment. Mycoviruses infection is a causative agent of mushroom strain degeneration.