In vitro Mutation of Chrysanthemum (Dendranthema grandiflora Tzvelev) with Ethylmethanesulphonate (EMS) in Immature Floral Pedicels

Many new cultivars of chrysanthemum were derived from induced mutation. However, chemical mutagenic agents are not widely used due to their low penetration into plant tissues. The objective of the present study was to induce mutation in chrysanthemum using ethylmethanesulphonate (EMS) in immature floral pedicels, followed by the induction and production of adventitious buds in vitro. Preliminary studies on the sensitivity of pedicels to EMS revealed that the LD50 was close to 0.82% (v/v). Immature pedicels of chrysanthemum cv. Ingrid (dark pink color) were treated with 0.77% (0.075 M) EMS solution for 1 h and 45 min, which was followed by rinsing in water for 15 min and surface disinfection. Afterwards, they were cultivated in Murashige and Skoog medium (salts and vitamins) amended with 1 g l⁻¹ of hydrolyzed casein, 1 mg l⁻¹ 6-benzylaminopurine (BAP) and 2 mg l⁻¹ indole-3-acetic acid (IAA). A total of 910 plants were obtained from the pedicels treated with EMS and were evaluated at the flowering stage. Forty-eight mutants (5.2%) were obtained, deviating in petal color (pink-salmon, light-pink, bronze, white, yellow and salmon color). Most of them (89.6% of the total) were phenotypically uniform. The results showed the efficiency of EMS to induce in vitro mutation of chrysanthemum.     

Effect of Manganese on Lignin Degradation by Pleurotus ostreatus during Solid-State Fermentation

Lignin degradation by Pleurotus ostreatus was studied under solid-state fermentation (SSF) in chemically defined medium containing various levels of Mn. Degradation of [¹⁴C]lignin prepared from cotton branches to soluble products, as well as its mineralization to ¹⁴CO₂, was enhanced by the addition of Mn. The effect of malonate on lignin mineralization was most marked during the first 10 days of SSF, in a treatment amended with 73 μM Mn. A high concentration of Mn (4.5 mM) caused inhibition of both fungal growth and mineralization rates during the first 2 weeks of incubation. Addition of malonate reversed this effect because of chelation of Mn. Mn was found to precipitate in all treatments, with or without the addition of malonate. α-Keto-γ-methiolbutyric acid cleavage to ethylene, an indication of ^. OH production, was observed as early as 3 days of incubation in all treatments.     

Shoot regeneration from stem and leaf explants of Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.)

Adventitious shoots were regenerated from leaf and stem explants of eleven chrysanthemum cultivars. The optimum medium for both explant types contained Murashige & Skoog basal medium supplemented with 5 M 6-benzylaminopurine and 5 M -napthaleneacetic acid. Generally, stem explants were superior to leaf explants. There were large cultivar differences in shoot regeneration frequency with three cultivars failing to respond over a wide range of hormone combinations. Shoots on stem explants appeared mainly to originate from cortical cells which rapidly divided and ruptured the epidermis. Regenerated shoots could be easily rooted, transferred to glasshouse conditions, and grown to flowering. All regenerated plants had the same morphological characteristics compared to plants derived from nodes.Supported in part by the National Biotechnology Program Research Grants Scheme in collaboration with Calgene Pacific P/L, Victoria, Australia.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

平菇(Pleurotus ostreatus)对稻草中木质素的生物降解及降解产物分析

变色圈试验证明平菇可以选择性优先降解稻草中的木质素,培养15d后,平菇对稻草中的木质素降解率为17.86%,对综纤维素降解率为2.44%,选择性指数为9.79。生料栽培平菇后,稻草中的木质素被降解50.24%。用气—质色谱(GC/MS)和红外光谱(IR)对木质素降解产物分析结果表明,平菇对稻草中木质素的降解效果十分明显,降解产物中检测出了大量含有苯环的小分子,证明木质素聚合体的降解首先发生在单体的侧链及单体间的连键上,发生Cα-Cβ、β-O-4等断裂,形成了单体。在进一步的降解过程中,平菇表现了其自身特有的降解机制,取代苯环单体上的甲氧基为甲基,而后发生苯环的开裂,这与报道的白腐菌降解过程有所不同。红外光谱分析中,平菇对木质素的降解明显,降解产物中含有很多木质素单体所特有的基团,如紫丁香基、愈创木基等,说明木质素的降解首先发生的是侧链的氧化反应。     

Leaf senescence in a non-yellowing cultivar of chrysanthemum (Dendranthema grandiflora)

Chlorophyll (Chl) and total soluble protein decreased and proteolytic activity increased over a 12-day period during dark-induced senescence in detached leaves of Tara, a yellowing cultivar (Y) of Dendranthema grandiflora . In Boaldi, a non-yellowing cultivar (NY), Chl and soluble protein remained near initial levels and little change in proteolytic activity was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins showed no major differences in banding patterns between the two cultivars at day 0; however, all of the resolved proteins were diminished in Tara by day 12. On the other hand, in NY Boaldi, the intensity of the protein bands did not change over the 12-day period. Attached and detached leaves exhibited similar senescence patterns for each cultivar. Ethylene (100 μl l⁻¹) accelerated the rate of Chl loss in detached leaves of Tara, but had no effect on Boaldi. These observations suggest that Boaldi is a stay-green genotype, possibly a functional type. The results are discussed in relation to the role of ethylene in chrysanthemum leaf senescence.     

Chemical control of plant habitus in summer-to-autumn flowering chrysanthemum (Dendranthema grandiflora)

Summer-to-autumn flowering chrysanthemums (Dendranthema grandiflora Tzvelev. cv.) are grown outdoors in containers and are characterised by a uniform ball-shaped growth habit. Development of new cultivars expanded the production of marketable plants from end of July until end of October. During the first weeks of the production cycle plants were pinched regular to increase branching but also to prevent development of precocious initiated flowers. This pinching treatment however increases Labour costs and is therefore more and more abandoned. The effectiveness of ethephon for terminal bud destruction was evaluated for three cultivars 'Draga', 'Tardero' and 'Veria Dark'. PLants were sprayed with 0-240-480-1200-2400 mg a.i./l. Ethephon spraying resulted in growth arrest and apical bud necrosis and higher doses resulted in more pronounced reactions. The highest dose (2400 mg a.i./l) resulted in phytotoxicity one week after application; phytotoxicity was more severe in 'Veria Dark' than 'Draga' and 'Tardero'. Ethephon treatments significantly delayed flowering and have potential to be used in commercial production schemes of summer-to-autumn flowering chrysanthemum in outdoor conditions.     

Lovastatin production by Pleurotus ostreatus: effects of the C:N ratio

The types of carbon source and nitrogen source used as well as the C:N ratio in the medium influenced lovastatin production by Pleurotus ostreatus. The maximum value of the lovastatin yield was obtained in a medium that contained organic nitrogen.     

The mannitol cycle in Pleurotus ostreatus (Florida)

In mushroom, presence of the mannitol cycle has not been reported so far although the polyol is supposed to be generated by the reduction of fructose by mannitol dehydrogenase. This study submits evidence for the presence of the mannitol cycle in Pleurotus ostreatus. The key enzyme of the cycle, mannitol-1-phosphate dehydrogenase (M1PDH), was present appreciably in all the developmental stages of the mushroom. However, the enzyme level dropped significantly at the onset of sporulation. The presence of M1DPH was confirmed by isozyme analysis and RT-PCR mediated amplification of a approximately 400 bp DNA fragment.     

Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye

The white-rot fungus Pleurotus ostreatus strain 32 is an excellent producer of the industrially important enzyme laccase. Laccase was the only ligninolytic activity detected in the supernatant when the fungus was grown in liquid culture with or without shaking. Growth and laccase production in static cultivation were superior to that in agitated cultivation, and N-limited culture is of benefit to laccase production. When using cellobiose and peptone as carbon and nitrogen source, a higher activity level was obtained. 2,2′-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) (1 mM) was shown to be the best inducer of laccase production, reaching maximum values of about 400 U/ml. Cu²⁺ (1 mM) also had a positive effect on laccase production, activity being enhanced to 360 U/ml. In addition, anthraquinone dye SN4R can be effectively decolorized by crude laccase (30 U/ml), the rate of which was 66%. The decolorization rate was increased by 90% with ABTS (0.16%) addition as a mediator of laccase.     

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.     

Biodegradation of Nigerian wood wastes by Pleurotus tuber-regium (Fries) Singer

Studies were carried out for 90days on the degradation of wood wastes of four economically important Nigerian trees; Terminalia superba, Mansonia altissima, Holoptelia grandis and Milicia excelsa by white rot fungus, Pleurotus tuber-regium a Nigerian edible mushroom. The pH of the wastes dropped to 4.0/4.2, 90days after incubation. On the contrary, amino nitrogen content of the wastes increased consistently during this period of solid-state fermentation. Lignin degradation also increased with the increase in incubation days. The greatest lignin reduction was observed in H. grandis followed by T. superba, M. altissima and M. excelsa. Digestibility of spent substrates by ruminants increased during fermentation as follows: M. excelsa>M. altissima>T. superba>H. grandis. These results are discussed in relation to the use of fermented wood wastes as feeds for ruminants.     

Alternative medium for production of Pleurotus ostreatus biomass and potential antitumor polysaccharides

Pleurotus species are recognized for producing beta-glucans with important medicinal properties as a constituent of the cellular wall of the fruiting body or of the mycelium. The aims of this work were to select a culture medium that maximized the production of biomass and polysaccharides produced by Pleurotus ostreatus DSM 1833 and to evaluate the selected medium in two values of initial oxygen transfer rate -K(L)a (10.2 and 19.3 h(-1)). A 2* *4 factorial design was constructed to evaluate the supplementation of wheat extract with corn steep liquor--CSL (10 or 20 g L(-1)), yeast extract--YE (2 or 5gL(-1)), ammonium sulfate--AS (0 or 5 g L(-1)) and glucose (20 or 40 g L(-1)). In terms of maximum productivity in biomass and global productivity in polysaccharides, the best values were obtained when 5 g L(-1) of YE and 40 g L(-1) of glucose were used. In terms of maximum concentration of biomass, the best results were obtained when 20 g L(-1) of CSL and 40 g L(-1) of glucose were used. The best results in terms of production of biomass and polysaccharides were achieved when lower initial K(L)a (10.2 h(-1)) was used.     

Biodegradation of endocrine-disrupting phthalates by Pleurotus ostreatus

Biodegradation of endocrine-disrupting phthalates [diethyl phthalate (DEP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP)] was investigated with 10 white rot fungi isolated in Korea. When the fungal mycelia were added together with 100 mg/l of phthalate into yeast extract-malt extract-glucose (YMG) medium, Pleurotus ostreatus, Irpex lacteus, Polyporus brumalis, Merulius tremellosus, Trametes versicolor, and T. versicolor MrP1 and MrP13 (transformant of the Mn-repressed peroxidase gene of T. versicolor) could remove almost all of the 3 kinds of phthalates within 12 days of incubation. When the phthalates were added to 5-day pregrown fungal cultures, most fungi except I. lacteus showed the increased removal of the phthalates compared with those of the nonpregrown cultures. In both culture conditions, P. ostreatus showed the highest degradation rates for the 3 phthalates tested. BBP was degraded with the highest rates among the 3 phthalates by all fungal strains. Only 14.9% of 100 mg/l BBP was degraded by the supernatant of P. ostreatus culture in YMG medium in 4 days of incubation, but the washed or homogenized mycelium of P. ostreatus could remove 100% of BBP within 2 days even in distilled water, indicating that the initial BBP biodegradation by P. ostreatus may be attributed to mycelium-associated enzymes rather than extracellular enzymes. The biodegradation rate of BBP by the immobilized cells of P. ostreatus was almost the same as that in the suspended culture. The estrogenic activity of 100 mg/l DMP decreased during biodegradation by P. ostreatus.     

Use of coffee grounds for production of Pleurotus ostreatus (Jacq.:Fr.) Kummer

Studies were carried out to screen the industrial strain HK35 of Pleurotus ostreatus for its ability to develop fruiting bodies in solid state cultivation using several substrates containing 17.8 to 55% coffee grounds. Our results showed that only 55% of coffee grounds was used in the substrate without detecting changes in fruiting body or on its biological efficiency of production. The chemical analysis of the caffeine in the substrate showed that this compound decreased about 59% of the mycelium activity, and no caffeine was found in fruiting bodies indicating its degradation by the fungal strain tested.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

The use of spent brewery grains for Pleurotus ostreatus cultivation and enzyme production

In the brewing industry, spent brewery grains (SBGs) are byproducts with a low economic value. The potential use of this leftover as a substrate ingredient for Pleurotus ostreatus fruiting body cultivation and enzyme production was evaluated. The best substrate mixture for P. ostreatus mycelium growth comprised 30% wheat bran (WB), 68% beech sawdust (BS) and 2% CaCO3. On the substrates containing SBG, the fastest mycelium growth was observed on the substrate composed of 10% SBG, 20% WB, 68% BS and 2% CaCO3. The highest biological efficiency (51%) of fruiting bodies was determined on the mixtures containing 20% WB, 10% SBG and 2% CaCO3. The SBGs with the addition of WB were also shown to be suitable as a substrate for enzyme production. However, the supplementation levels designate which enzymes are produced and in what amounts.     

Atrazine,desethylatrazine (DEA) and desisopropylatrazine (DIA) degradation by Pleurotus ostreatus INCQS 40310

Abstract

Atrazine is the most common herbicide applied in crops of economic relevance, such as sugar cane, soybean, and corn. Atrazine and its derivatives desethylatrazine (DEA) and desisopropylatrazine (DIA) are toxic to the environment, affecting animal and human health. Thus, this study aimed to evaluate the degradation of atrazine and its derivatives by the fungus Pleurotus ostreatus INCQS 40310, as well as the potential of the enzymes involved in this process. P. ostreatus INCQS 40310 was able to degrade atrazine (82%), DEA (71%), and DIA (56%) over 22?days of fungal cultivation. Proteomic analysis indicated the participation of hydrolases and peroxidases during the degradation process. Additionally, resting cells of the fungus were tested to verify the action of intracellular enzymes in the degradation process, suggesting the participation of cytochrome P450 enzymatic complex. Resting cells experiments promoted the degradation of 50% of atrazine, 36% of DIA, 30% of DEA. So far, this is the first work evaluating the biodegradation of DEA and DIA by fungus.     

The mitochondrial genome of the Basidiomycete fungus Pleurotus ostreatus (oyster mushroom)

In this study, the full mitochondrial genome of a basidiomycete fungus, Pleurotus ostreatus, was sequenced and analyzed. It is a circular DNA molecule of 73 242 bp and contains 44 known genes encoding 18 proteins and 26 RNA genes. The protein-coding genes include 14 common mitochondrial genes, one ribosomal small subunit protein 3 gene, one RNA polymerase gene and two DNA polymerase genes. In addition, one RNA and one DNA polymerase genes were identified in a mitochondrial plasmid. These two genes show relatively low similarities to their homologs in the mitochondrial genome but they are nearly identical to the known mitochondrial plasmid genes from another Pleurotus ostreatus strain. This suggests that the plasmid may mediate the horizontal gene transfer of the DNA and RNA polymerase genes into mitochondrial genome, and such a transfer may be an ancient event. Phylogenetic analysis based on the cox1 ORFs verified the traditional classification of Pleurotus ostreatus among fungi. However, the discordances were observed in the phylogenetic trees based on the six cox1 intronic ORFs of Pleurotus ostreatus and their homologs in other species, suggesting that these intronic ORFs are foreign DNA sequences obtained through HGT. In summary, this analysis provides valuable information towards the understanding of the evolution of fungal mtDNA.