An Improved Growth Medium to Assess Mycelial Compatibility Groups in Sclerotium rolfsii

Mycelial compatibility is assayed mainly by pairing mycelial plugs of field isolates on Petri dishes with agar media. Although methodologically simple, mycelial compatibility testing requires an artificial growth medium that permits the identification of compatible and incompatible interactions. In this work, several growth media were studied to assess consistently mycelial interactions between Sclerotium rolfsii isolates. A modification of Patterson’s medium with an increment of 25% glucose from the original concentration at a rate of 23.4 g/l and amended with 180 μl/l of red food colouring was the most effective combination for enhancing the size, density and distinctiveness of the aversion zone between incompatible isolates. This medium allowed the unequivocal identification of compatible and incompatible reactions of a set of five S. rolfsii isolates, which could be determined quickly after 5 days of incubation in the dark at 25°C. This new formulation improved significantly and consistently the assessment of the aversion zone reaction that was visible as a red line on the colony reverse as compared to that assessed using previous media formulations, for which the visualization of aversion zones was scarcely discernible. The utility of the improved growth medium was validated by microscopic observations of the contact area of hyphal pairings between isolates of S. rolfsii in microscope slide cultures.     

A high-affinity Zn2+ uptake system controls growth and biosynthesis of an extracellular,branched β-1,3-β-1,6-glucan inSclerotium rolfsii ATCC 15205

The plant pathogenic fungus imperfectusSclerotium rolfsii ATCC 15025 requires Zn²⁺ for both growth and biosynthesis of an extracellular, branched β-1,3-β-1,6-glucan in a completely defined mineral medium. Upon rising the external zinc concentration an increased yield of glucan inversely proportional to the yield of biomass was found in the cultivations with the bioreactor, but not in those with shake flasks. The complete carbon balance presented includes oxalic acid as an additional metabolite, secreted in rather high amounts due to the reduced oxygen supply in the viscous culture suspension. Only low amounts of zinc were accumulated. The successful development of an assay for the uptake of⁶⁵Zn²⁺ by homogeneous suspensions of zinc-depleted cultures ofS. rolfsii is reported. An energy-dependent highly specific Zn²⁺ uptake system, sufficient for growth and glucan synthesis, but no efflux system was demonstrated inS. rolfsii.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Degradation of oxalic acid (OA) producing Sclerotium rolfsii (Sacc.) by organic biocides

The aim of the study for the importance of oxalic acid produced by Sclerotium rolfsii during the invasion of host tissue during pathogenesis acts synergistically with endopolygalacturonase, lowering the pH of the infected tissues to a level optimal for the activity of this enzyme. Oxalic acid was the principal toxic agent produced in the culture filtrates of S. rolfsii and it was responsible for the death of host cells. The calcium present in structural pectates can be strongly chelated by oxalic acid. As a consequence, plant tissues are rendered more susceptible to invasion by S. rolfsii. Oxalic acid produced by S. rolfsii was very much reduced by Trichoderma viride (TVB1) (0.79 mg/ml culture filtrate), Pseudomonas fluorescens (SBHRPF2) 0.93 mg, neem cake (10%) (0.87 mg/ml), Lippia nodiflora (10%) and Lantana camera (10%) which were recorded as 2.12 and 2.25 mg/ml. The organic biocides that interfered with S. rolfsii led to reduction of oxalic acid production, which specifically reduced the disease incidence, and the oxalic acid degradation was a useful approach as a disease control strategy.     

Production of endopolygalacturonase fromRhizopus nigricans related to the evolution of growth substrate

Summary Endopolygalacturonases production byRhizopus nigricans is studied. The enzymatic level present during the early growth stages depolymerized fully the substrate and afterwards the growth of the fungi, with the parallel production of endopolygalacturonase, began. The maximum level of enzymatic activity was 18 U/mL and was reached six days after the maximun metabolic activity of the fungi. The production of endopolygalacturonase finished when the carbon source was exhausted.     

Inhibitory effect of methyl jasmonate on development of phytopathogen <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissl. and its reversal by ethephon and ACC

Methyl jasmonate (MeJA) was found to reduce spore germination, hyphal and mycelial growth in Alternaria alternata (Fr.) Keissl. The addition of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene precursor, together with MeJA to the culture medium resulted in a promotion of all developmental stages of the fungus; these compounds partially or completely reversed the inhibition due to MeJA depending on the concentrations applied. MeJA alone had no effect on ethylene production by mycelium, but after 6 days of incubation in the presence of ACC, emanation of this gas increased significantly. Ethylene is involved in reversing the inhibition of A. alternata due to MeJA.     

Enhancement of <Emphasis Type="Italic">Trichoderma</Emphasis> endochitinase secretion in tobacco cell cultures using an <Emphasis Type="Italic">α-</Emphasis>amylase signal peptide

An α-amylase signal peptide from rice was synthesized and fused with endochitinase (ech42) gene cloned from Trichoderma virens. The chimeric gene was designated as PSPα-amyech42, and this was transferred to a plant transformation vector, referred to as pMASGK. Leaf explants of tobacco cv White Burley were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying ech42 with its own signal peptide(ech42SP) and PSPα-amyech42(pMASGK) separately. Putative transformants were selected on Murashige and Skoog (MS) medium, supplemented with 1 mg/l bezyladenine (BA), 0.5 mg/l naphthalene acetic acid(NAA), and containing 200 mg/l kanamycin and 200 mg/l cefotaxime. Transformation was further confirmed by PCR with specific primers and Southern blot hybridization. Endochitinase secretion was quantified in 1-week-old cell suspension cultures obtained from 3-week-old callus cultures of transformants carrying PSPα-amyech42, transformants with ech42 and of control (untransformed) plant. Callus cultures of PSPα-amyech42 showed higher endochitinase activity (9–12 times) than those carrying ech42SP (7–8 times) in both medium and cell extracts. Media collected (200 μg of total protein) from PSPα-amyech42 suspension cultures in Potato Dextrose Agar plates showed growth inhibition of 73 and 53% against Sclerotium rolfsii and Rhizoctonia bataticola, respectively, whereas media collected (200 μg of total protein) from ech42SP suspension culture showed inhibition of 14 and 24% against Sclerotium rolfsii and Rhizoctonia bataticola, respectively.     

Use of Phanerochaete chrysosporium Immobilized on Kissiris for Synthetic Dye Decolourization: Involvement of Manganese Peroxidase

The mineral Kissiris, which is formed from the thickened foam of volcanic lava, was tested to approximate its mineral composition using energy-dispersive X-ray (EDX) analysis. The solid mineral contains silicon dioxide at about 16 (w/w). The considerable surface roughness of Kissiris along with its extensive porosity made this natural solid cell support an attractive candidate for manganese peroxidase (MnP) production for synthetic dye decolourization, at low cost. The white rot fungus Phanerochaete chrysosporium immobilized on the mineral Kissiris was grown in both stationary and agitated cultures (rotary shaker, 100 rev/min) using either carbon- or nitrogen-limited growth medium to study the ability of the fungus to degrade the synthetic dye methylene blue (MB). The value of residual dye for MB used at 60 ppm was 6% within 8 days of the incubation of the nitrogen-limited culture under the shaken conditions. Production of (MnP) occurred simultaneously in nitrogen-limited culture medium with the added MnSO₄ at 100 ppm. The MnP activity was at relatively high level (170 U/l).     

Serial cultivation of normal human keratinocytes: A defined system for studying the regulation of growth and differentiation

Summary We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologically differentiated epidermis.     

Improvement of artemisinin accumulation in hairy root cultures of Artemisia annua L by fungal elicitor

The effect of fungal elicitor, derived from mycelial extracts of Penicillium chysogenum 3446, on artemisinin production in hairy root cultures of Artemisia annua L was studied. Various concentrations of elicitor were added to the culture medium after 18 days. Time course experiments were carried out using a defined concentration of elicitor after 18 days. Various ages of hairy root cultures were elicited using a defined concentration of elicitor for 3 days. Artemisinin production in 21-day hairy root cultures treated with 0.3 mg total sugar/ml medium elicitor for 3 days reached to 549.1 mg/l.     

Toxicity of arsenic during high temperature bioleaching of gold-bearing arsenical pyrite

 A moderately thermophilic mixed culture, MT, and the thermophilic Sulfolobus acidocaldarius strain BC were studied for their response to arsenic in a defined medium and also in media containing a pyrite and an arsenical pyrite flotation concentrate. In defined medium, the individual constituents of the MT culture exhibited a high tolerance to arsenite and arsenate compared to S. acidocaldarius strain BC. When grown on increasing concentrations of the pyrite flotation concentrate, both cultures had similar specific leaching rates over the various concentrations of the mineral substrate. In contrast, S. acidocaldarius strain BC exhibited a decreasing specific leaching rate when grown on the arsenical pyrite while the MT culture was not affected. In addition, arsenic added to cultures of S. acidocaldarius strain BC growing with pyrite as a growth substrate inhibited further growth, while added arsenic had no effect on the MT culture growing on the pyrite. These data indicate that the moderately thermophilic, arsenic-resistant MT culture was able to leach arsenical pyrite more efficiently than was the S. acidocaldarius strain BC culture at high concentrations of the mineral. This emphasizes the fact that proper culture selection is an important parameter when developing commercial processes involving arsenic-containing minerals. Received: 21 June 1995/Received revision: 25 August 1995/Accepted: 7 September 1995     

Biodegradability of lignosulphonate by Streptomyces viridosporus strain T7A and a mixed natural microbial population antagonistic effects

The ability of a mixed natural microbial population, collected in an aerated lagoon treating Fluff pulp effluent and Streptomyces viridosporus strain T7A, to degrade lignosulphonate was evaluated. S. viridosporus growing in a mineral medium containing glycerol (7 g/l) and lignosulphonate (1 g/l) allowed 20% of lignosulphonate to be degraded after 18 days of incubation. A culture of the mixed population on culture medium after S. viridosporus growth was unable to degrade lignosulphonate products. Moreover, antagonism between S. viridosporus and the mixed population or between S. viridosporus and the isolated strains from this population was observed. The enhancement of lignosulphonate biodegradation by naturally occurring microorganisms in association with S. viridosporus (bioaugmentation strategy) seems to be difficult.     

Effect of perfluorodecalin as an oxygen carrier on actinorhodin production by Streptomyces coelicolor A3(2)

Perfluorodecalin, a perfluorocarbon (PFC), was used in this investigation as a dissolved oxygen carrier in the media of Streptomyces coelicolor cultures. The effects of different concentrations of PFC, PFC emulsified with pluronic F-68 and pluronic alone were investigated in the shake-flask cultures using both defined and complex media. In the defined medium with PFC alone, the maximum biomass and actinorhodin concentrations and the volumetric substrate consumption rates increased with increasing PFC concentration. They decreased dramatically, however, when the PFC concentration exceeded 50% (v/v). Emulsifying the PFC with pluronic F-68 resulted in a significant increase in antibiotic concentration while growth was unaffected. The inclusion of more than 4 g/l pluronic alone in the fermentation medium inhibited the growth. In the complex medium with 40% (v/v) PFC, although the final antibiotic concentration was unaffected, the onset of actinorhodin accumulation was 2 days earlier than that in the control. It was demonstrated that PFC and emulsified PFC did not have any deleterious effects on S. coelicolor cultures.     

Greening and growth of suspension-cultured cells of Chenopodium rubrum under conditions of heterotrophic and autotrophic nutrition

Abstract Dark-grown cell suspension cultures of Chenopodium rubrum lacking chlorophyll greened strongly upon transfer to illumination and fresh medium. This greening took place both in the presence of sucrose as a carbon source and in a mineral salt medium under an atmosphere enriched in CO₂. The synthesis of chlorophyll was in each case closely accompanied by the development of high levels of enzymes typical of photosynthesis. Greening in sugar-containing medium resulted in a rapid acquisition of characteristic features of photomixotrophic cultures, which have the ability to survive for a prolonged period in a minimal photoautotrophic environment in the light long after the initially present sucrose has been depleted from the medium. Greening under autotrophic conditions represented a direct transition from starvation conditions resulting from prolonged heterotrophic batch growth to successful photoautotrophy. Thus, light triggered the build-up of a competent photosynthetic apparatus irrespective of the nutritional necessity for autotrophy. Illumination and greening did not influence catabolic enzyme activities beyond that increase of metabolic activity which is required for the production of photosynthetic machinery.     

Ethylene Production and Mycelium Growth of the Tulip Strain of Fusarium oxysporum as Influenced by Shaking of and Oxygen Supply to the Culture Medium

The fungus Fusarium oxysporum Schlecht f. sp. tulipae Apt. can produce ethylene abundantly in vitro when grown in Pratt's liquid medium with glucose as the only organic substrate. This production starts after a lag phase of about 4 days, and peak production occurs when mycelium weight has reached its maximum value. For several days the rate of production is more or less linearly dependent on pO₂. The total production is also dependent on the oxygen concentration, but pure oxygen inhibits the total production by about 50% as compared with 21% oxygen. The high production in shake cultures, as compared with the low production in stagnant cultures, is probably the result of a better oxygen supply in the culture medium. The mycelium weight proved not to be a valid referential basis for the production of ethylene.     

Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze

Rosmarinic acid, an important phenolic active compound, is one of the main active constituents of Agastache rugosa Kuntze and has astringent properties, antioxidant capacity, anti-inflammatory activity, antimutagenic ability, antimicrobial capacity, and antiviral properties. To investigate in vitro production of rosmarinic acid, we established a hairy root culture of A. rugosa by infecting leaf and stem explants with Agrobacterium rhizogenes R1000, and tested the growth and rosmarinic acid production of these cultures. Hairy roots were cultured in Murashige and Skoog liquid medium and maximum growth (14.1 g dry wt/l) was attained after 14 days of culture, at which time the content of rosmarinic acid was 116 mg/g dry wt. The present results demonstrate that hairy root culture of A. rugosa is a valuable alternative approach for the production of rosmarinic acid.     

Changes in water potential and its components during shoot formation in tobacco callus

Addition of plant growth regulators (5 nM NAA and 5μM BAP) to a defined basal medium stimulated adventitious bud formation of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) cotyledon explants in culture. Cytoplasmic soluble proteins synthesized during early stages of adventitious bud formation were analyzed by electrophoresis of ³H- and ¹⁴C-leucine labeled proteins on SDS polyacrylamide gels. Increased synthesis of low molecular weight proteins (16,000 to 20,000 daltons) was detected after 2 days in culture and reached a maximal level at day 4. When cotyledon explants cultured on bud medium for 2 days were transferred to callus medium (which suppressed adventitious bud formation), suppression of the synthesis of low molecular weight proteins was also observed, suggesting that these proteins may be associated with early stages of adventitious bud formation.     

Medium-term preservation of mesophyll cells isolated from Asparagus officinalis L.: Development of a simple method by storage at reduced temperature

Simple conditions have been investigated allowing Asparagus officinalis L. mesophyll cells to be stored as high density suspensions with unimpaired growth capacities in cell cultures. In a classic cell culture medium, storage by itself affected the mesophyll cell population by a temperature-dependent increase of mortality. Long storage reduced the ability of living cells to enter the first mitosis when used as inoculum. This loss of mitotic capacity led to reduced growth capacities (i.e. reduced increase of the number of cells) of the cultures and was relieved by decreasing the temperature of storage. Storage at 4°C in a sucrose solution and in the usual Asparagus cell culture medium has been compared. The cell culture medium containing salts, sucrose and the hormones NAA, 6-BAP gave the best results. The effective period of cell preservation is defined as the period during which cultures could be obtained with recovery of mitotic activity and growth equaling at least 75% of the control. Even in the best conditions tested, this appeared variable and dependent upon the physiological age of the cladophylls from which the cells had been obtained. It was never less than 15 days and rose to 45 days when the cells came from young cladophylls.     

Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor

Parameters of VERO cell growth and metabolism were studied in cultures performed on microcarriers (MCs) using a bioreactor with a working capacity of 3.7?l. Kinetic studies of VERO cell growth in batch, semi-batch and perfusion cultures using concentrations of 2 and 10?mg/ml of MCs showed that a high concentration of MCs (10?mg/ml) and the use of medium perfusion allowed the attainment of higher final yields of VERO cells (6?×?10⁶ cells/ml after 10 days of culture). Perfusion also allowed better use of MCs as indicated by the observation of about 100% of MCs totally covered by cells and the appearance of multilayered cells on 64% of MCs after 13 days of VERO cell culture with 2?mg/ml of MCs. Concerning the concentration of nutrients in the cultures, the medium perfusion was able to sustain suitable levels of galactose and glutamine, which quickly decreased after 4 days in batch cultures. The air inlet in the batch cultures was capable of eliminating the NH₄ ⁺ which accumulated in the medium culture. Lactate accumulated during the first days of culture but then was utilized by the cells and decreased along the culture time. The optimization of VERO cell cultures on microcarriers as indicated by the concentration of MCs, medium perfusion and air inlet is discussed.     

Increased production of laccase by <Emphasis Type="Italic">Cerrena unicolor</Emphasis> in submerged liquid cultures

The wood-degrading basidiomycete Cerrena unicolor C-139 has been suggested as a potential producer of the industrially important enzyme laccase. Basic culture parameters influencing the enzyme synthesis in shaken-flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably enhanced by the addition of Cu²⁺ in the micromolar range to a carbon-sufficient and nitrogen-sufficient culture medium (C/N = 16.69). When an optimised medium containing glucose (10 g/L) and l-asparagine (1.5 g/L) was used, and enzyme synthesis was stimulated by addition of 10 μM Cu²⁺ to the culture medium on days 3, 6 and 9, maximal laccase productivity obtained after 17 days’ cultivation in shaken flask cultures was above 100,000 nkat/L. In fermenter fungal cultures, the influence of stabilisation of medium pH on laccase activity was additionally studied. The use of a bioreactor with an automatic pH control set at pH 6.5 after 48-h incubation resulted in the enzyme activity of 65,000 nkat/L after 8 days’ cultivation.