A mathematical model applied to the fungal colony growth of Sclerotium rolfsii

Colonies of Sclerotium rolfsii growing on solid medium showed a linear increase in radius simultaneously with exponential changes in colonial weight. The mathematical model developed by Koch (Koch, A.L. 1975. J. Gen. Microbiol. 89, 209216) to describe similar kinetics of mycelial growth was applied. Most of the parameters defined in the mentioned model were directly measured or calculated, except the values of K (maximum mycelium carrying capacity) and S (initial mycelial density) which were fitted to the experimental data of growth at 30°C and 23°C. The ratios K/S at 30°C and at 23°C were 18.8 and 1200 respectively, being consistent with two-dimensional growth. The model was predictive only during the exponential increase of the colony weight.     

Enzymatic hydrolysis of cellulosic materials by Sclerotium rolfsii culture filtrate for sugar production.

The hydrolysis of purified celluloses (cotton, Avicel, Cellulose-123, Solka Floc SW40) and cellulosic wastes (rice straw, sugarcane bagasse, wood powders, paper factory effluents) by Sclerotium rolfsii CPC 142 culture filtrate was studied. Factors which effect saccharification such as pH, temperature, enzyme concentration, substrate concentration, produce inhibition, adsorption, and inactivation of enzyme and particle size were studied. Virtually no inhibition (less than 3%) of cellulose hydrolysis by the culture filtrate was observed by cellobiose and glucose up to 100 mg/mL. Filter paper degrading enzyme(s) (but neither carboxymethylcellulase nor beta-glucosidase) was adsorbed on cellulose. The n value in the S. rolfsii system was calculated to be 0.32 for Avicel P.H. 101 and 0.53 for alkali-treated (AT) rice straw indicating penetration of cellulase into AT rice straw. In batch experiments at 10% substrate level, solutions containing 6 to 7%, 3.8 to 4.7%, 4.0 to 5.1%, and 4.2 to 4.9% reducing sugars were produced in 24 to 48 from AT rice straw. AT bagasse, alkali - peracetic acid treated mesta wood and paper factory sedimented sludge effluent, respectively. The main constituent in the hydrolysate from cellulose was glucose with little or no cellobiose, probably due to the high cellobiase content in the culture filtrate.     

Some chemical and biochemical changes in straw constituents during growth of Pleurotus flabellatus (Berk & Br) Sacc

Summary The quantitative changes in constituents of rice straw during different stages of growth of the fungus Pleurotus flabellatus were investigated. Cellulose, hemicellulose(s), lignin, total carbon and total nitrogen showed a continuous decrease from inoculation until the end of fruit body harvesting, whereas free sugars, total ash and C/N ratio increased. As calculated on constant ash basis, 14 and 13.9% of cellulose, 6.6 and 7% of hemicellulose(s) and 4 and 1.5% of lignin were decomposed during the mycelial growth and fructification respectively. Total N decreased by 0.16 and 0.23% during the mycelial growth and fructification respectively. The progressive breakdown of cellulose and hemicellulose(s) was correlated to an apparent increase in the activities of celullase and hemicellulase(s). The trend in development of cellulases and -glucosidase activities in the substrate during different stages of its growth was demonstrated.     

Xylanase formation by Sclerotium rolfsii: effect of growth substrates and development of a culture medium using statistically designed experiments

A culture medium for increased xylanase formation by a wild strain of Sclerotium rolfsii was optimized by applying statistically designed experiments. The optimization process was divided into three basic steps. In the initial phase of screening, two different fractional factorial plans, a Graeco-Latin square design and a folded Plackett-Burman design, were employed. From the list of medium components the relevant variables for xylanase formation (cellulose, peptone, and trace elements) were identified. The second step of optimization used a central composite experimental design to calculate a predictive model. In this phase only the two most important factors, i. e. cellulose and peptone from meat, were considered. The third step of verification validated the results of the optimization. Optimal concentrations of cellulose and peptone were found to be 42.6 and 80.0 g 1^–1, respectively. Shaken flask cultivations of S. rolfsii using the optimized medium yielded a maximum xylanase activity of 394 IU ml^–1 (6,570 nkat ml^–1) within 13 days. Growth on the optimized medium also resulted in elevated levels of other hemicellulolytic enzyme activities including mannanase, -arabinosidase, and acetyl esterase. Especially the value of 155 IU · ml^–1 (2,580 nkat ml^–1) for mannanase is remarkable since it appears that this is among the highest activities reported for fungal organisms.Dedicated to Prof. Dr. Robert M. Lafferty on the occasia of his 65th birthday     

Extracellular enzyme production during anamorphic growth in the edible mushroom,Pleurotus sajor-caju

Cultivation of mushrooms on lignocellulosic wastes represents a cost-effective organic recycling process. Pleurotus sajor-caju grown on cotton-waste produced relatively low levels of three components of the cellulase complex namely cellobiohydrolase (EC 3.2.1.91), CMCase (EC 3.2.1.4) and -glucosidase (EC 3.2.1.21) with specific activity values of 10.0, 71.4 and 21.6U (mg protein)–1 respectively after 15days. Higher specific activity was registered in alkali-treated cotton with 15.6, 83.4 and 56.1U (mg protein)–1 respectively after 20days. Lower levels were noted on rubber-tree sawdust substrate with specific activity values of 0.28, 0.62 and 0.75U (mg protein)–1 for the respective enzymes after 28–35days growth. The maximum production of xylanase (EC 3.2.1.8) of 0.63U (mg protein)–1 occurred after 20days while a relatively higher level of the phenoloxidase enzyme, laccase (EC 1.14.18.1) of 27.4U (mg protein)–1 (maximum) was found after 35days. Laccase, the activity of which is associated with morphogenesis, increased with mycelial growth, peaked at maximum growth and thereafter decreased rapidly. This could prove important commercially in timing the end of spawn-run in preparation for initiation of fruiting.     

The effects of light and tyrosinase during sclerotium development in Sclerotium rolfsii Sacc.

Some effects of light on morphogenesis in Sclerotium rolfsii Sacc. were studied. Physiological competence to visible light developed during the first 120 h after inoculation, with an optimum sensitivity phase between 84 and 96 h that coincided with the leading hyphae reaching the edge of the Petri dish. Although sclerotial initials were produced in dark-grown cultures, light was necessary for the continuation of the developmental and maturation phases of sclerotial morphogenesis. Tyrosinase activity (o-diphenol: oxygen oxidoreductase, EC 1.10.3.1) was detected during sclerotial formation and the pH and temperature optima for his polyphenol oxidase in vitro were about 6.0 and 45 degrees C respectively. The enzyme was inhibited by cysteine. Similar activity levels of tyrosinase were obtained in blue and "white" light-grown cultures but in red light activity was comparable with that of dark-grown cultures. Laccase activity was not detected at any stage of development.     

Determining biomass in viscous culture broths of Sclerotium rolfsii during β-glucan production: hexosamine assay as an indirect alternative technique

Micro-Kjeldahl determination of protein content in the cells was not suitable for the determination of biomass because of the changing composition of the cells. The significant correlation was found during cultivation between the values of the hexosamine assay and those corresponding to the dry weight method (r = 0.98, slope = 1.04) which showed the validity of both hexosamine and dry weight methods, with the former being a simple, reliable and not time consuming alternative for the biomass estimation of Sclerotium rolfsii during scleroglucan production.     

Vermicompost-based bioformulation for the management of sugarbeet root rot caused by Sclerotium rolfsii

Vermicompost-based bioformulations of bacterial and fungal biocontrol agents were examined against sugarbeet root rot caused by Sclerotium rolfsii. The result showed that the Pseudomonas fluorescens strain Pf1 in combination with either Trichoderma asperellum strain TTH1 or Bacillus subtilis strain EPCO-16 performed better in reducing disease next to the chemical difenoconazole. Similarly, enhanced yield was observed in the same combination treatments under both pot and field conditions.     

Effect of Pseudomonas sp. P7014 on the growth of edible mushroom Pleurotus eryngii in bottle culture for commercial production

Addition of bacterial culture strain P7014 and its supernatant to the mushroom growing media resulted in mushroom mycelia run faster. Mycelial growth rate of Pleurotus eryngii was increased up to 1.6 fold and primordial formation was induced one day earlier. Moreover, it was supposed that addition of bacteria had beneficial applications for commercial mushroom production, which appreciably reduced total number of days for cultivation of about 5+/-2 days compared with uninoculated, which took 55+/-2 days.     

齐整小核菌内源小核菌胶水解酶的挖掘及其功能验证

小核菌胶是一种高分子水溶性微生物胞外多糖,主要用于石油开采、食品加工、医药制造和化妆品等领域.由微生物发酵法生产的小核菌胶分子量较高,存在溶解性低、粘度高和分散性差等问题,对提取、保存和应用带来了一系列困难.研究用于改性小核菌胶分子量的降解方式对扩大小核菌胶的工业应用具有重要意义.文中以齐整小核菌Sclerotium ...     

Isolation and characterization of glyoxylate dehydrogenase from the fungus Sclerotium rolfsii.

Glyoxylate dehydrogenase (glyoxylate:NAD+ oxidoreductase) was purified 600-fold in three steps from crude extracts of the fungus Sclerotium rolfsii (Corticium rolfsii Curzi). Two of the purification steps involved dye-affinity chromatography. The enzyme is a tetramer of Mr 250 000, with identical subunits of Mr 57 000. Inhibition studies suggest that there is one essential thiol group per active site.     

Use of complex media for the production of scleroglucan by Sclerotium rolfsii MTCC 2156

Submerged fermentation was carried out for the production of scleroglucan by Sclerotium rolfsii MTCC 2156 using complex media, such as coconut water, sugarcane molasses and sugarcane juice at 28+/-2 degrees C and 180 rpm for 72 h. Sugarcane juice gave maximum scleroglucan production of 23.87 g/l as compared to 12.58 and 18.45 g/l with coconut water and sugarcane molasses, respectively. Utilization of these substrates would be ecologically sound and economically advantageous.     

用转基因棉籽作平菇培养料时平菇主要害虫的发生动态

为了评估转基因棉籽对平菇害虫发生的影响,利用转Bt基因棉籽及其亲本对照棉籽配制平菇培养的菌棒,在菇棚中自然感虫,观察了平菇厉眼菌蚊、粪蚊、蚤蝇和螨类的发生情况。结果表明,含对照棉籽菌棒中平菇厉眼菌蚊和螨类发生量高于含转基因棉籽菌棒, 随着棉籽浓度的增加,对照棉和转Bt基因棉对平菇厉眼菌蚊均表现出抑制作用。对照棉籽对粪蚊的抑制作用比转Bt基因棉籽强,但随着棉籽浓度的增加,含对照棉籽菌棒中粪蚊发生量呈递增趋势。随着棉籽浓度的增加,含对照棉籽菌棒中蚤蝇发生量呈递增趋势,含转Bt基因棉籽菌棒中蚤蝇发生量递减;低含量时对照棉籽对蚤蝇的抑制作用强,而高含量时转Bt基因棉籽对蚤蝇的抑制作用强。     

Evaluation of host and pathogen responses for screening soil amendments to control Sclerotium rolfsii

Three strains of Bradyrhizobium, 280A, 2209A and 32H1, that nodulated peanuts (Arachis hypogaea L.), were tested for their ability to grow and survive at elevated temperatures of up to 42°C in laboratory culture. Strain 32H1 was unable to grow at 37°C and was more sensitive to elevated temperatures than the other two strains. All three produced heat-shock proteins of molecular weights 17 kDa and 18 kDa. Two greenhouse experiments were conducted to determine the effect of high root temperature on nodulation, growth and nitrogen fixation of peanut. Two peanut varieties (Virginia cv NC7 and Spanish cv Pronto) were inoculated and exposed to root temperatures of 30°, 37° and 40°C. Nodulation and nitrogen fixation were strongly affected by root temperature but there was no variety × temperature interaction. At a constant 40°C root temperature no nodules were formed. Nodules were formed when roots were exposed to this temperature with diurnal cycling but no nitrogen fixation occurred. Highest plant dry weight, shoot nitrogen content and total nitrogen were observed at a constant root temperature of 30°C. Increasing root temperature to 37°C reduced average nitrogen content by 37% and total nitrogen by 49% but did not reduce nodulation. The symbiotic performance of the strains corresponded to their abilities to grow and survive at high temperature in culture.     

Modeling and scale-up of the unsterile scleroglucan production process with Sclerotium rolfsii ATCC 15205

An empirical model was applied to describe the growth related formation of scleroglucan in batchwise cultivation of Sclerotium rolfsii. In this case, the level of oxygen supply controls the carbon flux into glucan, biomass, and CO₂ evolution and therefore determines the yield coefficients Y_Glucan/BDM and Y_BDM/O2. It was observed that scleroglucan formation is enhanced under microaerobic conditions. However, as the empirical model and data of actual batch cultivations show, different maxima exist for product end concentration [g/l] and volumetric productivity [g/ld] depending on the total oxygen uptake during cultivation. A sufficient bulk mixing of the highly viscous culture suspension becomes particularly important during large-scale cultivations. In addition, the scleroglucan production process proved to be shear sensitive. A correlation between the attainable molecular weight of the glucan and the stirrer tip velocity in bioreactors of different sizes is presented. For all these reasons, a scale-up of this process is very complex. Large-scale cultivations under microaerobic conditions, aiming for maximum product end concentration, were slowed down by poor bulk mixing leading to a lower carbon flux into glucan formation. On the other hand, a scale-up designed for maximum volumetric productivity using high oxygen supply was successfully conducted up to a reactor volume of 1.500 l. To minimize the loss in product quality (molecular weight of the glucan) due to high stirrer tip velocities, a mixing concept was developed employing reduced agitation combined with maximum aeration to secure a sufficient axial bulk mixing in the reactor.     

Effect of Moisture and Temperature on the Survival of Sclerotia of Sclerotium rolfsii in Soil

The sclerotia of Sclerotium rolfsii Sacc. survived in natural soil for 225 days under controlled moisture at 50% water holding capacity (WHC) after which there was a progressive reduction in the population of viable sclerotia. At 390 days only 48% were recovered. Sclerotia survived well at moisture contents upto 75% WHC but at 100% the population declined rapidly and none were recovered after 60 days. The contents of the sclerotia were found to lyse without germination leaving hollow rinds. Such lysis was found to be favoured between 25 and 40°C. At and below 20°C no such lysis was recovered and more than 80% sclerotia were recovered even after 60 days.     

Anti-fungal activity of essential oils of Ceylon Eucalyptus species for the control of Fusarium solani and Sclerotium rolfsii

The essential oils were extracted from the leaves of Eucalyptus microcorys, Eucalyptus grandis and Eucalyptus robusta which were grown in Sri Lanka and their major chemical compounds were determined. 1,8-Cineole and α-pinene were identified as major aroma compounds in these oils. In this study, the anti-fungal activity of essential oils of E. microcorys, E. grandis and E. robusta, ethanol extract of E. microcorys and 1,8-cineole were evaluated against Sclerotium rolfsii, a fungi responsible for leaf spot disease of indoor plants and Fusarium solani, a fungi responsible for dry rot diseases of potato by poisoned food technique, and minimum inhibitory concentrations (MICs) were determined. The essential oils from three Eucalyptus species showed significant inhibitory effect against S. rolfsii and F. solani than the ethanol extract of E. microcorys. Of treatments, the essential oil of E. grandis showed the best anti-fungal activity with the MIC values of less than 0.1% for S. rolfsii and 0.5% for F. solani. The MICs of the oils of E. microcorys and E. robusta were between 0.3–0.5% against S. rolfsii and 0.5–0.75% for F. solani. The 1,8-cineole did not exhibit inhibition activity as much of Eucalyptus essential oils and hence, it can be assumed that minor chemical components of the oils contribute to the growth inhibition of the tested fungi. This is the first report of anti-fungal activity of Sri Lankan oils of E. microcorys, E. grandis and E. robusta and ethanol extract of E. microcorys against S. rolfsii and F. solani. These findings would be useful for the designing of natural fungicide for agriculture- and food-based industries.     

Purification and Characterization of Cellobiose Dehydrogenase from the Plant Pathogen Sclerotium (Athelia) rolfsii

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, β-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.