High temperature enhances the ability of Trichoderma asperellum to infect Pleurotus ostreatus mycelia.
ABSTRACT: Trichoderma asperellum is one of the species which can be isolated from contaminated Pleurotus ostreatus cultivation substrate with green mold disease. This study focused on the relationship between high temperature and infectivity of T. asperellum to P. ostreatus. Antagonism experiments between T. asperellum and P. ostreatus mycelia revealed that high temperature-treated P. ostreatus mycelia were more easily infected by T. asperellum and covered by conidia. Microscopic observation also showed that P. ostreatus mycelia treated with high temperature could adsorb more T. asperellum conidia. Furthermore, conidia obtained from T. asperellum mycelia grown at 36°C featured higher germination rate compared with that incubated at 28°C. High temperature-treated T. asperellum mycelia can produce conidia in shorter periods, and T. asperellum mycelia were less sensitive to high temperature than P. ostreatus. Deactivated P. ostreatus mycelia can induce T. asperellum cell wall-degrading enzymes (CWDEs) and P. ostreatus mycelia subjected to high temperature showed induced CWDEs more effective than those incubated at 28°C. Moreover, T. asperellum showed higher CWDEs activity at high temperature. In dual cultures, hydrogen peroxide (H2O2) increased after 36°C, and high concentration of H2O2 could significantly inhibit the growth of P. ostreatus mycelia. In summary, our findings indicated for the first time that high temperature can induce a series of mechanisms to enhance infection abilities of T. asperellum to P. ostreatus mycelia and to cause Pleurotus green mold disease.
Project description:Pleurotus ostreatus is widely cultivated in China. However, its cultivation is strongly affected by seasonal temperature changes, especially the high temperatures of summer. Nitric oxide (NO) was previously reported to alleviate oxidative damage to mycelia by regulating trehalose. In this study, we found that NO alleviated oxidative damage to P. ostreatus mycelia by inhibiting the protein and gene expression of aconitase (ACO), and additional studies found that the overexpression and interference of aco could affect the content of citric acid (CA). Furthermore, the addition of exogenous CA can induce alternative oxidase (aox) gene expression under heat stress, reduce the content of H2O2 in mycelium, and consequently protect the mycelia under heat stress. An additional analysis focused on the function of the aox gene in the heat stress response of mycelia. The results show that the colony diameter of the aox overexpression (OE-aox) strains was significantly larger than that of the wild-type (WT) strain under heat stress (32°C). In addition, the mycelia of OE-aox strains showed significantly enhanced tolerance to H2O2 In conclusion, this study demonstrates that NO can affect CA accumulation by regulating aco gene and ACO protein expression and that CA can induce aox gene expression and thereby be a response to heat stress.IMPORTANCE Heat stress is one of the abiotic stresses that affect the growth and development of edible fungi. Our previous study found that exogenous NO had a protective effect on mycelia under heat stress. However, its regulatory mechanism had not been elucidated. In this study, we found that NO altered the respiratory pathway of mycelia under heat stress by regulating aco The results have enhanced our understanding of NO signaling pathways in P. ostreatus.
Project description:Catalases are ubiquitous hydrogen peroxide-detoxifying enzymes. They participate in fungal growth and development, such as mycelial growth and cellular differentiation, and in protecting fungi from oxidative damage under stressful conditions. To investigate the potential functions of catalases in Pleurotus ostreatus, we obtained two catalase genes from a draft genome sequence of P. ostreatus, and cloned and characterized them (Po-cat1 and Po-cat2). Po-cat1 (group II) and Po-cat2 (group III) encoded putative peptides of 745 and 528 amino acids, respectively. Furthermore, the gene structures were variant between Po-cat1 and Po-cat2. Further research revealed that these two catalase genes have divergent expression patterns during different developmental stages. Po-cat1/Po-cat1 was at a barely detectable level in mycelia, accumulated gradually during reproductive growth, and was maximal in separated spores. But no catalase activity of Po-cat1 was detected by native-PAGE during any part of the developmental stages. In contrast, high Po-cat2/Po-cat2 expression and Po-cat2 activity found in mycelia were gradually lost during reproductive growth, and at a minimal level in separated spores. In addition, these two genes responded differentially under 32 °C and 40 °C heat stresses. Po-cat1 was up-regulated under both temperature conditions, while Po-cat2 was up-regulated at 32 °C but down-regulated at 40 °C. The accumulation of catalase proteins correlated with gene expression. These results indicate that the two divergent catalases in P. ostreatus may play different roles during development and under heat stress.
Project description:Heat stress seriously threatens the growth of Pleurotus ostreatus. Various studies have been performed to study the resistance of P. ostreatus to heat stress. Here, the metabolome was evaluated to determine the response of P. ostreatus mycelia to heat stress at different times (6, 12, 24, 48 h). More than 70 differential metabolites were detected and enriched in their metabolic pathways. Dynamic metabolites changes in enrichment pathways under heat stress showed that heat stress enhanced the degradation of unsaturated fatty acids and nucleotides, increased the content of amino acids and vitamins, and accelerated glycolysis and the tricarboxylic acid cycle in P. ostreatus. The time course changes of P. ostreatus metabolites under continuous heat stress demonstrated that amino acids continuously changed with heat stress, nucleotides clearly changed with heat stress at 12 and 48 h, and lipids exhibited an increasing trend with prolonged heat stress, while few types saccharides and vitamins changed under heat stress. Additionally, heat-treated P. ostreatus produced salicylic acid and other stress-resistant substances that were reported in plants. This study first reported the metabolites changes in P. ostreatus mycelia during 48 h of heat stress. The metabolic pathways and substances that changed with heat stress in this research will aid future studies on the resistance of P. ostreatus and other edible fungi to heat stress.
Project description:The purpose of this study was to optimize the extraction method for polysaccharide from the fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm and to assess the antioxidant and cytotoxic potentials of polysaccharide. In this investigation, polysaccharides from Pleurotus ostreatus (Jacq.) P. Kumm were extricated by utilizing the hot water. One-single factor and response surface methodology was established to optimize the extraction conditions for polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm. Examination of antioxidant activity of Pleurotus ostreatus polysaccharide (POP) was directed by utilizing 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) techniques. Cytotoxicity of POP was evaluated using an MTT assay. The experimental data were fitted to a quadratic equation utilizing multiple regression investigations, and the ideal conditions were as per the following: water/crude material proportion, 26.04 mL/g; an extraction time of 62.08 minutes; and an extraction temperature 70.5°C. Under such conditions, the polysaccharide yield was 5.32 ± 0.12% with the anticipated yield. POP showed good scavenging activity against DPPH radical (p<0.001, EC50 = 1036.38 ?g/mL, R2 = 0.8313) and ABTS radicals (p<0.001, EC50 = 824.37 ?g/mL, R2 = 0.8223), with a dose (p<0.001)-and-time (p<0.001) dependent cytotoxic potential on Ehrlich ascites carcinoma cell line in vitro. This demonstrated that polysaccharides (POP) had certain cancer prevention agent exercises. In this manner, these examinations give reference to additionally research and reasonable improvement of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide and POP may prove a useful therapeutic agent, due to its robust antioxidant and cytotoxic activity.
Project description:Pleurotus ostreatus is a species of white-rot fungi that effectively degrades lignin. In this study, we aimed to efficiently express the lac-2 gene of Pleurotus ostreatus in the Pichia pastoris X33 yeast strain. The enzymatic properties of recombinant yeast were determined, and its ability to degrade corn stover lignin was determined. The results showed the optimum pH values of recombinant laccase for 2,2'-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid, 2,6-dimethoxyphenol, and 2-methoxyphenol were 3.0, 3.0, and 3.5, respectively. The optimum reaction temperature was 50 °C, and it had good thermal stability and acid and alkali resistance. The degradation rate of lignin in corn stover by recombinant laccase was 18.36%, and the native Pleurotus ostreatus degradation rate was 14.05%, the difference between them is significant (p < 0.05). This experiment lays a foundation for the study of the degradation mechanism of lignin by laccase.
Project description:Photoresponse mechanism of oyster muchroom mycelia was studied using a custom microarray prepared on the basis of the genome information (Pleurotus ostreatus PC15 v2.0) in JGI Genome Portal. Blue light stimulation to the mycelia caused the up-regulation and down-regulation of genes expression. Particulary, the genes coding rate-controlling enzymes in glycolysis, pentose phosphate, and sikimic acid pathways were up-regulated to accumulate shikimic acid dramatically. Overall design: In this study, the time course of gene expression in oyster mushroom mycelia caused by blue light stimulation using light emitting diodes (LED) was analyzed at 0, 0.5, 1, 3, 6, 12, 24, 36 h.
Project description:The aim of the study was to obtain new compounds during biotransformation of two halocompounds, the ?-bromo and ?-iodo-?-bicyclolactones 1 and 2. Unexpectedly Pleurotus ostreatus produced together with the hydroxylactone, 2-hydroxy-4,4-dimethyl-9-oxabicyclo[4.3.0]nonane-8-one (3), its own metabolite (3S,9S,15S)-(6E,12E)-3,9,15-trimethyl-4,10,16-trioxacyclohexa-deca-6,12-diene-1,5,8,11,14-pentaone (4). The method presented here, in which this macrosphelide 4 was obtained by biotransformation, has not been previously described in the literature. To the best of our knowledge, this compound has been prepared only by chemical synthesis to date. This is the first report on the possibility of the biosynthesis of this compound by the Pleurotus ostreatus strain. The conditions and factors, like temperature, salts, organic solvents, affecting the production of this macrosphelide by Pleurotus ostreatus strain were examined. The highest yield of macroshphelide production was noticed for halolactones, as well with iodide, bromide, iron and copper (2+) ions as inductors.
Project description:The worldwide commercial production of the oyster mushroom Pleurotus ostreatus is currently threatened by massive attacks of green mold disease. Using an integrated approach to species recognition comprising analyses of morphological and physiological characters and application of the genealogical concordance of multiple phylogenetic markers (internal transcribed spacer 1 [ITS1] and ITS2 sequences; partial sequences of tef1 and chi18-5), we determined that the causal agents of this disease were two genetically closely related, but phenotypically strongly different, species of Trichoderma, which have been recently described as Trichoderma pleurotum and Trichoderma pleuroticola. They belong to the Harzianum clade of Hypocrea/Trichoderma which also includes Trichoderma aggressivum, the causative agent of green mold disease of Agaricus. Both species have been found on cultivated Pleurotus and its substratum in Europe, Iran, and South Korea, but T. pleuroticola has also been isolated from soil and wood in Canada, the United States, Europe, Iran, and New Zealand. T. pleuroticola displays pachybasium-like morphological characteristics typical of its neighbors in the Harzianum clade, whereas T. pleurotum is characterized by a gliocladium-like conidiophore morphology which is uncharacteristic of the Harzianum clade. Phenotype MicroArrays revealed the generally impaired growth of T. pleurotum on numerous carbon sources readily assimilated by T. pleuroticola and T. aggressivum. In contrast, the Phenotype MicroArray profile of T. pleuroticola is very similar to that of T. aggressivum, which is suggestive of a close genetic relationship. In vitro confrontation reactions with Agaricus bisporus revealed that the antagonistic potential of the two new species against this mushroom is perhaps equal to T. aggressivum. The P. ostreatus confrontation assays showed that T. pleuroticola has the highest affinity to overgrow mushroom mycelium among the green mold species. We conclude that the evolutionary pathway of T. pleuroticola could be in parallel to other saprotrophic and mycoparasitic species from the Harzianum clade and that this species poses the highest infection risk for mushroom farms, whereas T. pleurotum could be specialized for an ecological niche connected to components of Pleurotus substrata in cultivation. A DNA BarCode for identification of these species based on ITS1 and ITS2 sequences has been provided and integrated in the main database for Hypocrea/Trichoderma (www.ISTH.info).
Project description:BACKGROUND:Phenylalanine ammonia-lyase (PAL, EC 184.108.40.206) is the first key enzyme in the phenylpropanoid pathway. The pal gene has been widely studied in plants and participates in plant growth, development and defense systems. However, in Pleurotus ostreatus, the biological functions of pal during organismal development and exposure to abiotic stress have not been reported. RESULTS:In this study, we cloned and characterized the pal1 (2232?bp) and pal2 (2244?bp) genes from the basidiomycete P. ostreatus CCMSSC 00389. The pal1 and pal2 genes are interrupted by 6 and 10 introns, respectively, and encode proteins of 743 and 747 amino acids, respectively. Furthermore, prokaryotic expression experiments showed that PAL enzymes catalyzed the conversion of L-phenylalanine to trans-cinnamic acid. The function of pal1 and pal2 was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the two pal genes had similar expression patterns during different developmental stages. The expression of pal genes was higher in the reproductive growth stage than in the vegetative growth stage. And the interference of pal1 and pal2 delayed the formation of primordia. The results of heat stress assays showed that the RNAi-pal1 strains had enhanced mycelial tolerance to high temperature, while the RNAi-pal2 strains had enhanced mycelial resistance to H2O2. CONCLUSIONS:These results indicate that two pal genes may play a similar role in the development of P. ostreatus fruiting bodies, but may alleviate stress through different regulatory pathways under heat stress.
Project description:Successive cultivation of fungi on artificial media has been reported to cause the sectorization, which leads to degeneration of developmental phenotype, and virulence. <i>Fusarium oxysporum</i> f. sp. <i>niveum</i> (Fon), the causal agent of watermelon Fusarium wilt, forms degenerated sectors after successive cultivation. In the present research, we demonstrated that subculture with aged mycelia increased the incidence of degenerations. To further investigate the differences between the Fon wild type (sporodochial type, ST) and variants (MT: mycelial type and PT: pionnotal type), developmental phenotypes and pathogenicity to watermelon were examined. Results in variants (PT2, PT3, PT11, and MT6) were different from ST with mycelia growth, conidia production and chlamydospore formation. Virulence of degenerated variants on susceptible watermelon Grand Baby (GB) cultivar was determined after inoculation with Fon variants and Fon ST. In root dipping methods, Fon variants showed no significant differences in disease progress compared with ST. Fon variants showed a significant decrease in disease progression compared with ST through infested soil inoculation. The contrasting results of two inoculation methods suggest that the degenerative changes due to repeated successive cultivation may lead to the loss of pathogen virulence-related factors of the early stage of Fon infection process. Therefore, cell wall-degrading enzymes (CWDEs; cellulase, pectinase, and xylanase) activities of different variants were analyzed. All Fon degenerated variants demonstrated significant decreases of CWDEs activities compared with ST. Additionally, transcript levels of 9 virulence-related genes (<i>fmk1</i>, <i>fgb1</i>, <i>pacC</i>, <i>xlnR</i>, <i>pl1</i>, <i>rho1</i>, <i>gas1</i>, <i>wc1</i>, and <i>fow1</i>) were assessed in normal state. The degenerated variants demonstrated a significantly low level of tested virulence-related gene transcripts except for <i>fmk1</i>, <i>xlnR,</i> and <i>fow1</i>. In summary, the degeneration of Fon is triggered with successive subculture through aged mycelia. The degeneration showed significant impacts on virulence to watermelon, which was correlated with the reduction of CWDEs activities and declining expression of a set of virulence-related genes.