Age Of Mycelium Research Articles

Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

Fatty acid composition in lipid fractions lengthwise the mycelium of Mortierella isabellina and lipid production by solid state fermentation

This paper investigates the correlation between mycelial age and fatty acid biosynthesis. The correlation was investigated by analyzing the lipid composition lengthwise the mycelium of the oleaginous fungus Mortierella isabellina, a potential producer of γ-linolenic acid (GLA). Young mycelia were rich in polar lipids (glycolipids plus sphingolipids and phospholipids), while neutral lipid content increased in aged mycelia. In young mycelia, each polar lipid fraction contained almost 40% (w/w) polyunsaturated fatty acids (PUFAs), but this content decreased to less than 30% (w/w) in aged mycelia. On the other hand, PUFA content in neutral lipids fluctuated slightly with age. These results indicate that PUFA biosynthesis is favored in young, fast growing mycelia, while it decreases significantly in aged mycelia. This trend was also observed when we grew M. isabellina on pear pomace, an agro-industrial waste. Pear pomace cultures yielded significant amounts of lipid, which reached 12% (w/w) in dry fermented mass. The produced lipid was rich in GLA and the maximum GLA content in dry fermented mass was 2.9 mg/g.

Optimization of different factors for efficient protoplast release from entomopathogenic fungusMetarhizium anisopliae

Optimization of different factors for efficient protoplast release fromMetarhizium anisopliae was investigated. Factors like culture media, age of the mycelium, incubation time, different enzymatic combinations and osmotic stabilizer were studied. Mycelium harvested at 40th h in Sabouraud Dextrose broth showed the best protoplast yield over the other media and age of mycelium tested. An incubation time of 3 h and Lysing enzyme at a concentration of 10 mg/ml was the best among the different enzymatic concentrations and combinations tested and yielded a protoplast release of 7.3×108 protoplasts/ml. The most suitable osmotic stabilizer for efficient protoplast release was 0.7M KCl.

Protoplast from β-carotene-producing fungus Blakeslea trispora: Preparation, regeneration and validation

The effects of key parameters on the preparation and regeneration of protoplast from the β-carotene-producing fungus Blakeslea trispora were discussed in this paper, including the combination of various enzymes, mycelial age, digesting time and temperature, pH value, osmotic stabilizers, pretreatment, culture medium and culture method. Under the condition of mixed enzymes in osmotic stabilizer (0.6 M NaCl) combined with 2% lysozyme, 3% cellulase and 3% snailase, the highest protoplast yield, as high as 7.48×106 protoplasts/mL, was obtained when mycelial age was 60 h at pH 5.0–6.0 with digesting for 14–16 h at 28 °C. After purification of the obtained protoplasts, they were regenerated in PDA regenerative medium using bilayer plate culture method. To validate the usability of the protoplasts, a novel plasmid with green fluorescent protein (GFP) was used in transformation for easy visual observation. The results showed that the protoplasts prepared by the optimized method were active and applicable in further gene manipulation experiments.

Obtenção de protoplastos do fungo filamentoso Aspergillus ochraceus

A obtenção de protoplastos é uma ferramenta importante para a transformação genética de fungos. Neste trabalho foi estudada a influência de fatores como idade micelial, tipo e concentração de enzimas e estabilizadores osmóticos na produção de protoplastos de Aspergillus ochraceus. Os melhores resultados de produção de protoplastos foram obtidos utilizando-se NH4Cl 0,8mol L-1 como estabilizador osmótico, micélio com 24h de crescimento e a combinação de Lysing Enzymes e Meicelase ambas, a 20mg mL-1. Entretanto, bons resultados foram também obtidos com a utilização apenas de Lysing Enzymes.

Decolorization of Cotton Pulp Black Liquor by Pleurotus ostreatus in a Bubble-column Reactor

Decolorization of cotton pulp black liquor by Pleurotus ostreatus B1 in a bubble-column reactor (BCR) was studied. The optimal conditions for the running of BCR are 30 degrees C, pH 6.0, aeration rate 1.2 L min(-1), and mycelial age 7 days. Under the optimal conditions, the BCR was run for four cycles (each cycle, 12 days) and the same mycelial pellets were reused. The ultimate decolorization and COD removal rates are 76% and 80%, respectively.

A study of steroid hydroxylation activity of Curvularia lunata mycelium

It has been demonstrated that the mycelium of Curvularia lunata at the end of the logarithmic growth phase displays a maximal 11-hydroxylase activity towards cortexolone (4-6 g/l) used for transformation as a microcrystalline suspension in phosphate buffer. The mycelium at a later stage of fungal growth displays an elevated 14-hydroxylase activity, necessary for generation of 14-hydroxyandrostenedione. The effects of different forms of substrate added to the reaction mixture, age and concentration of mycelium, and fungal clones tolerant to salts of heavy metals (0.35-0.5%) were studied to remove the side 14-hydroxylation, accompanying the main cortexolone transformation. Mycelia of the fungal clones tolerant to Co2+ and Cu2+ displayed a weak hydroxylase activity or its complete absence and an elevated content of melanin, the biosynthesis of which is intensified under adverse conditions. The results obtained suggest that the transformation of steroids by the studied C. lunata strain is a detoxication of foreign compounds.

Transport of ergot alkaloids and quinocitrinins in the producing fungus Penicillium citrinum

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to absorb autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to absorb ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12- day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.

Decolourisation of reactive textile dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by Funalia trogii ATCC 200800

Decolourisation of reactive dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by white rot fungi Funalia trogii was studied under static conditions. The effect of various conditions such as mycelial age, initial dye and glucose concentrations on decolourisation were also investigated. Decolourisation activity of F. trogii was compared with Phanerochaete chrysosporium known as test microorganism. It was found that 7-day-old cultures were more effective than 5-day-old cultures of F. trogii for decolourisation of these dyes. Decolourisations by F. trogii of both dyes were increased with glucose concentration decreasing. In contrast, decolourisations by P. chrysosporium were decreased. F. trogii decolourised 92-98% of both dyes within 4-10 h. However, P. chrysosporium partiallydecolourised (11-20%) these dyes during 10 days incubation period under the same conditions.

Selected cultural and environmental parameters influence disease severity of dandelion caused by the potential bioherbicidal fungi, Phoma herbarum and Phoma exigua

Selected cultural and environmental variables were investigated for their influence on the efficacy of Phoma herbarum and Phoma exigua to cause disease on dandelion (Taraxacum officinale) under growth room conditions. In both species, mycelial fragments caused significantly greater disease severity on dandelion than spore suspensions. Mycelial age was not an important factor in disease severity caused by P. herbarum, with all cultures causing high disease ratings. However, younger cultures of P. exigua caused the greatest disease severity on dandelion, but significantly less than that caused by P. herbarum. The initial pH of the growth medium (potato dextrose broth) did not affect disease severity caused by either Phoma species. Increasing concentrations of mycelia of P. herbarum were applied to dandelions that were then exposed to various leaf wetness durations. Disease severity increased with increasing leaf wetness duration. For dandelions exposed to no leaf wetness duration, the greater the mycelial concentration, the greater the disease rating. However, for dandelions exposed to all leaf wetness durations, all concentrations of mycelia caused similar disease ratings. As P. herbarum caused high disease ratings on dandelion, it therefore warrants further investigation as a potential bioherbicide for the control of this weed.

Decolourisation of Reactive Black 5 by Funalia trogii immobilised on Luffa cylindrica sponge

The decolourisation of Reactive Black 5 (RB5) by immobilised Funalia trogii was investigated. Cultures of F. trogii immobilised on Luffa cylindrica sponge could effectively decolourise the dye. The effect of mycelial age was also studied, and decolourisation rate of a 3-day-old age culture was higher (8.22 mg dye/g dmw day) than those of 0- and 6-day-old cultures (6.86 and 7.80 mg dye/g dmw day). Macroscopic and microscopic examinations showed that dye was not biosorbed on the fungal mycelium. The growth of F. trogii was inhibited by all tested dye concentrations with compared to controls but this effect was minimised when the fungus was completely immobilised on the sponge. Using optimal mycelial age, cultures of L. cylindrica sponge were tested for their ability for dye decolourisation at different initial concentration. The kinetic parameters of decolourisation were calculated according to Lineweaver–Burk plots ( K m of 106.04 mg dye/l and V max of 117.64 mg dye/l day).

Transformation of Fusarium oxysporum f. sp. cubense, causal agent of Fusarium wilt of banana, with the green fluorescent protein (GFP) gene

Fusarium oxysporum f. sp. cubense (Foe) is the causal agent of Fusarium wilt (Panama disease) of bananas in most tropical and subtropical banana-producing regions of the world. The fungus infects through roots, colonises the rhizomes and eventually blocks the vascular system of the pseudostems, resulting in plant death. The green fluorescent protein (GFP) emits green fluorescence when excited by blue light, making it a useful tool to study early stages of fungal infection. The objective of this study was to transform Foc isolates with the GFP gene. Isolates representing ‘subtropical’ race 4 of the fungus were transformed with the sGFP derivative using hygromycin as a selectable marker. Efficiency and transformation of spheroplasts depended on mycelium age, the choice of enzymes and the temperature and duration of incubation. The transformed isolates did not differ markedly from the wild type isolates in growth and morphological characteristics in vitro. Fluorescence microscopy showed expression of the green fluorescent protein in fungal structures. The presence of the GFP DNA in the fungal cells was confirmed by PCR using a GFP-specific primer pair and Southern blot analysis. Pathogenicity tests showed that the transformation process did not alter pathogenicity of Foc isolates. Fungal hyphae within tissues of infected plants could be seen to fluoresce and the transformed fungus was re-isolated from artificially inoculated plants. Transformants of Foc will facilitate future infection studies with this pathogen on banana.

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126.

Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.

Statistical analysis on some critical parameters affecting the formation of protoplasts from the mycelium of Penicillium griseofulvum

The critical parameters, such as slant age, mycelium age and contact time of mycelium with lytic enzymes which affects the formation of protoplast from the Penicillium griseofulvum mycelium were studied using a novel technique called response surface methodology. The design used to optimize these parameters was full factorial central composite design. The number of protoplasts, viability of protoplasts and griseofulvin yield are the three responses studied in this communication. The individual optimum values of slant age, inoculum age and contact time obtained are 187.1, 30.81, 21.4 h, respectively, for number of protoplast/ml 68.92, 33.01, 9.88 h, respectively, for percentage viability of protoplasts and 230.2, 73.6, 31.2 h, respectively, for griseofulvin. In order to obtain a single optimum value for all the three responses, a multi-response analysis was carried out using a generalized distance approach. The simultaneous optimum values of slant age, inoculum age and contact time are 133.78, 37.1 and 15.97 h, respectively, for all the three responses.

Protoplast preparation and transient transformation of Rhizoctonia solani

Optimization of protoplast release (202 × 10−4 ml−1) from a strain of Rhizoctonia solani anastomosis group three was achieved by treating 3 d old mycelia from broth cultures with Novozyme 234 for 3 h at 30°g; C. The only other treatment, of many tested, that yielded any protoplasts (15.6 × 10−4 ml−1) was commercial lysing enzymes from Trichoderma harzianum. Various combinations of commercial enzyme preparations, variations of osmotic stabilisers, and mycelial age, were ineffective in raising the protoplast yield. Regeneration of colonies from protoplasts was maximal (83-87%) when protoplasts were incorporated in cooled agar containing 5% glucose, malt extract and yeast extract, supplemented with 0.8 or 1.0 M mannitol as osmotic stabiliser. Liquid culture broth and other potential osmotic protectants at various concentrations yielded fewer regenerating colonies (5% in broth and 13-75% in agar). The Rhizoctonia strain was highly sensitive to hygromycin B, so transformation of protoplasts was achieved with a polyethylene glycol-based procedure, using plasmid pES200 containing the bacterial hygromycin B phosphotransferase gene driven by a trpC promoter from Aspergillus nidulans. Transformation was achieved using the DNase inhibitor aurintricarboxylic acid and incubation temperatures below 10-12°. It was confirmed by high stringency hybridization of DNA extracted from the transformants, using pES200 as a radiolabelled probe. The transformants grew slowly (25-35 mm diam after 14 d) then ceased growth, and never produced fast-growing sectors indicative of integrated plasmid DNA. Transformation was also achieved with pAXHY2 from Cryphonectria parasitica, and colonies reached 55-65 mm diam before they stopped growing. Southern blots confirmed the presence of non-integrated pAXHY2, but no fast-growing sectors were produced.

Possible relationship between aggressiveness of two <em>Eutypa lata</em> isolates and their polyamines and abscisic acid content

<p style="text-align: justify;">By HPLC analysis, we showed that myceliums of two <em>Eutypa lata</em> isolates cultivated in Enikson and Petersson medium culture synthesized polyamines (Putrescine, Spermidine and Spermine) and abscisic acid (ABA).</p><p style="text-align: justify;">ln this medium, myceliums of high virulent isolate (BXI-IO) produced fewer polyamines than less virulent isolate (BX 1-5) while inverse phenomenon was observed for ABA content. The two growth regulators were diffused in medium culture in which their levels increase with age of mycelium. In the end, variations of PA and ABA in myceliums show antagonist effect of the two growth regulators in <em>Eutypa lata</em> fungi.</p>

Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae

AbstractPreparation and regeneration of mycelial protoplasts from Alternaria eichhorniae were examined. A commercially available muralytic enzyme, Novozym 234, was used for isolation of protoplasts. The mycelial age and the pH of the stabilized buffer affected the formation of protoplasts. The maximum production of protoplasts (3,9 × 108/g fresh weight mycelia) was obtained from 24‐h‐old mycelia digested with Novozym 234 (20 mg/ml) in a stabilized buffer of pH 6.4 and incubated in the dark at 30°C on a rotary shaker (90 r.p.m.) for 6 h. Morphological characteristics of the protoplasts varied and depended on the age of the mycelia used in protoplast production. Moreover, mycelial age had a highly significant influence (P = 0.0001) on the frequency of protoplast regeneration.

Decolourisation of Orange II by a Wood-Rotting Fungus

The decolourisation of Orange II by a wood-rotting fungus has been studied. It was found that Fungus F29 could effectively decolourise Orange II especially when grown as pelleted mycelia under agitated conditions. Many factors affecting the decolourisation process in nitrogen-limited media (NLM) were studied, including: concentration of glucose, NH4+, Mn(II) and veratryl alcohol; initial pH; amount of mycelium; mycelial age; Orange II concentration; temperature. Results showed that the media containing Orange II at 1000 mg dm−3 (or higher) could be decolourised by 98% of the initial colour (A480 nm) in 2 days, in most conditions tested, and that the mycelia could be repeatedly reused. © 1997 SCI.

Development of a monoclonal antibody-based immunoassay for the detection and quantification of Anguillospora longissima colonizing leaf material.

Monoclonal antibodies (MAbs) to the aquatic hyphomycete Anguillospora longissima were raised in mice by using a coimmunization program. A cell line was raised that produced a MAb of the immunoglobulin M class that was specific for A. longissima both in an enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence but that did not recognize other members of the aquatic hyphomycetes. This MAb (AL-HH8c) was used to develop a quantitative ELISA in vitro. The antigen recognized by AL-HH8c is produced throughout the mycelium, irrespective of mycelial age and culture conditions. By using this MAb, mycelium of A. longissima colonizing leaf material can be detected by ELISA, immunofluorescence, and immunoenzymatic staining methods.

Lipid Production by Lipomyces lipoferus

AbstractLipomyces lipoferus was studied for its lipid production by investigating the inhibitory and stimulatory effects of different metals on lipid synthesis. The nature of metallic ions present in the medium exercise a considerable influence on the lipid storage. Chlorides of Ca++ and Mg++ ' show a marked inhibition on the formation of lipid materials while the acetates of the same metallic ions show improving effect on the amount of lipid produced. It is further noted that Na+ and K+ ions show better results for lipid production as compared to Ca++ and Mg++ when used as their acetates. The utilisation of sugar is also less effective with Ca++ than with Na++. The fatty acid composition of lipids extracted from cells of L. lipoferus shows that palmitic acid and oleic acid are the major components. However, the percentage of these acids increases with the age of mycelium by 11 % and 6% respectively. TLC results reveal that triglycerides are the major components of the neutral lipids (90 % ).