Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus

Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn(2+)-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn(2+)-dependent and Mn(2+)-independent peroxidase activity under Mn(2+)-deficient culture conditions.     

Cultivation of Pleurotus ostreatus on weed plants

Oyster mushroom, Pleurotus ostreatus (Jacq.:Fr.) Kumm. ITCC 3308 (collected from Indian Type Culture Collection, IARI, New Delhi, India, 110012) was grown on dry weed plants, Leonotis sp, Sida acuta, Parthenium argentatum, Ageratum conyzoides, Cassia sophera, Tephrosia purpurea and Lantana camara. Leonotis sp. was the best substrate in fruit body production of P. ostreatus when it was mixed with rice straw (1:1, wet wt/wet wt) for mushroom cultivation. The fruiting time for P. ostreatus was also less on Leonotis sp. than on any other weed substrates tested in the present investigation. T. purpurea was the least suited weed for oyster mushroom cultivation. The main problem of oyster mushroom cultivation on weed substrates was found to be low yield in the second flush that could be overcome by blending weed plants with rice straw. The protein contents of the fruit bodies obtained from Cassia sophera, Parthenium argentatum and Leonotis sp. were not only better than rice straw but also from the rice straw supplemented weeds.     

Isolation of genes expressed during the developmental stages of the oyster mushroom, Pleurotus ostreatus, using expressed sequence tags

In order to isolate and identify the developmentally regulated genes during fruiting body development, cDNA libraries were constructed from eight developmental stages of the Oyster mushroom, Pleurotus ostreatus. From these libraries, 11 761 expressed sequence tags (PoESTs) were generated. Of these, 4060 different genes (PoUnigenes) were identified, representing 34.5% of the entire genome. Redundancy analysis of ESTs during the developmental stages identified eight, 13 and two genes that were specifically expressed in mycelia, fruiting body and basidiospore, respectively. RT-PCR was used to confirm the specific expression of nine genes which showed specific redundancy in fruiting body stages, four genes of which were expressed specifically in fruiting body stages as expected in redundancy analysis, and other genes were expressed abundantly in fruiting body stages. The EST database of P. ostreatus generated during this study provides a genetic and biochemical basis for future studies of the developmental stages of basidiomycetes.     

Particle and naked RNA mycoviruses in industrially cultivated mushroom Pleurotus ostreatus in China

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 2.5kb 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.     

Isolation of a LIM15/DMC1 homolog from the basidiomycete Coprinus cinereus and its expression in relation to meiotic chromosome pairing

The Escherichia coli gene recA is essential for homologous recombination and DNA repair, and homologs have been identified in eukaryotes. A basidiomycete, Coprinus cinereus, which has many advantages for the study of meiosis, was recently reported to have a homolog of one of these, RAD51. In the yeast Saccharomyces, mutations in the RAD5I gene cause defects in both somatic and meiotic cells. Based on this finding, we screened for a meiosis-specific homolog of recA, equivalent to Lilium LIM15 or Saccharomyces DMC1, in C. cinereus, and isolated a clone containing a 1.2-kb DNA fragment from a cDNA library constructed with Coprinus poly(A)⁺ RNA isolated from cells undergoing meiosis. The predicted amino acid sequence was 52% identical to the putative gene product of the lily cDNA clone LIM15 and 61% identical to Saccharomyces DMC1, and showed limited sequence similarity to the products of RAD52, 55, and 57. The synchrony of meiosis in Coprinus provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. Northern analysis indicated that Coprinus LIM15/DMC1 was expressed at meiotic prophase within 8 h after the onset of karyogamy, suggesting that the gene functions mostly at the stage at which the homologous chromosomes pair, but may not be essential at the point at which they recombine. The gene is not expressed in somatic cells. Received: 8 October 1998 / Accepted: 22 July 1999     

Lignocellulolytic enzymes profile during growth and fruiting of Pleurotus ostreatus on wheat straw and tree leaves

Cultivation of two commercial Pleurotus ostreatus (oyster mushroom) strains was performed in plastic bags. Tree leaves appeared to be an excellent growth substrate for the conversion into fruiting bodies with biological efficiency of 108-118%. The level of enzyme activity was strongly regulated during the life cycle of mushrooms. However, despite the quantitative variations, each strain had a similar pattern of enzyme accumulation in fermentation of both substrates. Laccase and MnP activities were high during substrate colonization and declined rapidly during fruiting body development. On the contrary, in substrate colonization P. ostreatus expressed comparatively low activity of hydrolases. When primordia appeared, the activity of these enzymes sharply increased. Both cellulase and xylanase activity peaked at the mature fruiting body stage. When mushrooms shifted to the vegetative growth, the activity of ligninolytic enzymes again gradually increased, whereas the activity of hydrolases decreased.     

DNA marking of some quantitative trait loci in the cultivated edible mushroom Pleurotus ostratus (Fr.) Kumm

Fungi of the genus Pleurotus, in particular, species Pleurotus ostreatus (common oyster mushroom) are among most cultivated fungi in the world. Due to intense rates of development of studies in this field, efficient breeding programs are highly required in the search for new P. ostreatus strains. The principal traits used worldwide for selecting strains are intensity of fruitbearing, fruit body cap color (for some consumptive markets), and mycelium growth rate. In this connection, the objective of this work was to study these quantitative traits and to find molecular markers, which could be employed to accomplish breeding programs. In general, we found 12 genomic loci (quantitative trait loci, QTLs) controlling mycelium growth rate of oyster and six QTLs responsible for the fruit body cap color. The genetic map of P. ostreatus was constructed, and all markers of quantitative traits found by us were located on this genetic map. The obtained linkage map can be a useful tool for the accomplishment of breeding programs to improve economically important traits of oyster mushroom.     

Purification and characterization of a new ribonuclease from fruiting bodies of the oyster mushroom Pleurotus ostreatus.

A ribonuclease (RNase), possessing an N-terminal sequence disparate from those of ribonucleases from other mushrooms and previously isolated Pleuotus ostreatus RNases, was purified from the fruiting bodies of the edible mushroom Pleurotus ostreatus. The N-terminal sequence of Pleurotus ostreatus RNase did not manifest homology even to a previously reported RNase from the same mushroom. The ribonuclease was adsorbed on CM-Sepharose and Mono S. It exhibited a molecular mass of 12 kDa in both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease displayed an activity of 11490 U/mg on yeast tRNA. The highest ribonuclease activity was exhibited toward poly U, followed by poly A and poly C. No activity was shown toward poly G. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 240 nM.     

Purification, characterization, and cloning of fibrinolytic metalloprotease from Pleurotus ostreatus mycelia

A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and 35 degrees C, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the A alpha-chain and the B beta-chain over the gamma-chain. Enzyme activity was enhanced by the addition of Ca2+, Zn2+, and Mg2+ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis.     

Veratryl alcohol stimulates fruiting body formation in the oyster mushroom, Pleurotus ostreatus

The oyster mushroom, Pleurotus ostreatus, cultivated in solid state on sugarcane bagasse-wheat bran (5:1) medium in the presence of veratryl alcohol resulted in an increased production of the fruiting body at earlier times compared to when the fungus was grown in the absence of veratryl alcohol. The results indicate a new physiological role for veratryl alcohol in stimulating fruiting body formation. Veratryl alcohol also stimulated laccase production during the mycelial growth stage. Evidence is also presented that laccases were involved in the physiological development of the fruiting body.     

Identification of non-pseudomonad bacteria from fruit bodies of wild agaricales fungi that detoxify tolaasin produced by Pseudomonas tolaasii

Bacterial isolates from wild Agaricales fungi detoxified tolaasin, the inducer of brown blotch disease of cultivated mushrooms produced by Pseudomonas tolaasii. Mycetocola tolaasinivorans and Mycetocola lacteus were associated with fruit bodies of wild Pleurotus ostreatus and wild Lepista nuda, respectively. Tolaasin-detoxifying bacteria belonging to other genera were found in various wild mushrooms. An Acinetobacter sp. was isolated from fruit bodies of Tricholoma matsutake, Bacillus pumilus was isolated from Coprinus disseminatus, and Sphingobacterium multivorum was isolated from Clitocybe clavipes. A Pedobacter sp., which seemed not be identifiable as any known bacterial species, was isolated from a Clitocybe sp. Tolaasin-detoxifying bacteria identified thus far were attached to the surface of mycelia rather than residing within the fungal cells. M. tolaasinivorans, M. lacteus, B. pumilus, the Pedobacter sp., and S. multivorum efficiently detoxified tolaasin and strongly suppressed brown blotch development in cultivated P. ostreatus and Agaricus bisporus in vitro, but the Acinetobacter sp. did so less efficiently. These bacteria may be useful for the elucidation of mechanisms involved in tolaasin-detoxification, and may become biological control agents of mushroom disease.     

Influence of wheat cultivars on straw quality and Pleurotus ostreatus cultivation

The main raw material for Pleurotus ostreatus (oyster mushroom) cultivation is wheat straw. Estimation of straw biodegradability from 15 different spring wheat cultivars under irrigation in South Africa was determined using linear discriminant analysis to discriminate or group the 15 cultivars by combining chemical analysis and in vitro enzymatic hydrolysis. Significant differences (P < 0.01) were found between ash, nitrogen, reducing sugars, anthrone reactive-carbohydrates, water-soluble dry matter, and oyster mushroom yields. The significance of these measurements was investigated and discussed.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg²⁺ but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Cultivation of oyster mushroom on waste paper with some added supplementary materials

Spawn running, pin head and fruit body formation, and mushroom yield of oyster mushroom (Pleurotus ostreatus) on waste paper supplemented with peat, chicken manure and husk rice (90+10; 80+20 w:w) were studied. The fastest spawn running (mycelia development) (15.8 days), pin head formation (21.4 days) and fruit body formation (25.6 days), and the highest yield (350.2 gr) were realized with the substrate composed of 20% rice husk in weight. In general, increasing the ratio of rice husk within the substrate accelerated spawn running, pin head and fruit body formation, and resulted in increased mushroom yields, while more peat and chicken manure had a negative effect on growing.     

Lipid content and fatty acid composition of the higher edible fungus--the oyster mushroom Pleurotus ostreatus (Fr.) Kummer

The content of free lipids in wild and cultivated plant bodies, as well as in the surface and deep mycelium of the higher edible basidiomycete Pleurotus ostreatus (Fr.) Kummer--Oyster mushroom--was studied. By means of GL chromatography fatty acids with the number of C atoms from 8 to 20 were identified. In plant bodies and mycelium of the Oyster mushroom unsaturated fatty acids preveil; the main portion falls to oleic acid (up to 56%).     

启动子替代构建糙皮侧耳漆酶高产菌株

POXC是糙皮侧耳合成最多的一种漆酶。应用启动子替代技术,用构巢曲霉的甘油醛-3-磷酸脱氢酶基因（gpd）启动子替代POXC基因的启动子,构建了超量表达POXC糙皮侧耳转化子。转化子中POXC基因表达量比出发菌株提高了0.72–3倍。在PDA平板培养、PD摇瓶培养和棉籽壳试管培养条件下,转化子漆酶活力显著提高,比出发菌株提高了1.5倍以上。用棉籽壳栽培,转化子菇产量比出发菌株提高了16.2%,培养料中木质素含量比出发菌株减少21%。结果表明,应用高效启动子替代能够显著提高糙皮侧耳漆酶基因的表达量、漆酶活力及其木质纤维素降解能力。     

伞菌DNA简易序列分析引出的思考

目的:伞菌物种以子实体形态特征为分类依据,研究以菌丝体代替子实体进行种质资源鉴定的遗传证据。方法:以常见的食用伞菌香菇、平菇子实体的不同部位组织及其分离菌丝体为供试材料,制备了12个随机引物介导的RAPD-PCR指纹,把DNA指纹图谱转化为简易的序列数据,经生物信息处理软件比较分析。结果:香菇不同菌株子实体DNA相似性系数在0.886～0.986之间,平菇不同菌株子实体DNA相似系数在0.779～0.976之间。对于供试的单个子实体而言,香菇和平菇子实体的菌盖、菌褶、菌柄及其组织分离菌丝体,与以此为菌种栽培得到的子实体相比较,所获得的有限DNA指纹图谱全部相同。结论:揭示了香菇和平菇不同菌株的遗传多样性,初步反映了伞菌不同发育阶段在分子水平上的遗传变异和亲缘关系,争论了以菌丝体代替子实体使用RAPD手段进行种质资源鉴定与系统发育分析引出的真菌遗传问题。