Influence of humidity on production of pelleted fungal inoculum

One of the practical problems in scaling-up the production of fungal inocula for environmental applications is how to provide essential humidity for fungal growth. Pelleted solid substrate was used as a fungal biomass carrier. It was coated with alginate or agar hydrogels that contained mycelial fragments of the white-rot fungi Trametes versicolor or Irpex lacteus. To follow fungal growth and formation of mycelial coat over pelleted substrate, the fluorescein-diacetate hydrolysing activity (FDA) assay and visual inspection were used. Both fungi were able to overgrow the pelleted substrate in 5–6 days, at a relative humidity (RH) of 86.3% or higher. The enrichment of alginate hydrogel with nutrients or coating of pelleted substrate with more hydrophilic agar hydrogel enabled I. lacteus to overgrow the pellets at a lower RH of 83.6%. Fungal inocula produced at lower RH possessed lower final moisture contents and had greater mechanical strength. Conditioning of T. versicolor mycelial fragments, by a 3-h incubation in fresh growth medium, enhanced fungal growth over the pelleted substrate. A mathematical model was used to simulate and to explain moisture distribution in a hydrogel-coated pellet and the formation of mycelial coat, for various conditions of fungal inocula production.     

Correlation of mycelial growth rate with other phenotypic characters in evolved genotypes of Aspergillus nidulans

Fungal populations can adapt to their environment by the generation and fixation of spontaneous beneficial mutations. In this study we examined whether adaptation, measured as an increased mycelial growth rate, has correlated responses in the filamentous fungus Aspergillus nidulans with several other metric characters that could be important fitness components (colony forming units, germination speed, and biomass formation). Studying 60 populations that had evolved over 800 generations by experimental evolution, we find that only mycelial growth rate increased during adaptation to growing on solid medium. We further found that among evolved strains colony forming units is negatively correlated with mycelial growth rate and that colony forming units and biomass formation show a positive correlation. Our results give insight into changes in fungal phenotype as a result of adaptation and suggest that mycelial growth rate is the only available target of selection.     

Dibutyryl c-AMP as an inducer of sporidia formation: Biochemical and antigenic changes during morphological differentiation of Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>) pathogen in axenic culture

Effect of dibutyryl adenosine 3′,5′-cyclic monophosphate (dbc-AMP), an analogue of c-AMP, was investigated on growth and morphological differentiation ofTilletia indica. Exponential growth was observed up to 21 days in both presence and absence of dbc-AMP; however, increasing concentration of dbc-AMP was deleterious to mycelial growth in liquid culture. A slow increase of mycelial biomass up to 21 days and decline at 30 days in the presence of 2.5 mM dbc-AMP was observed, therefore, this concentration was chosen in subsequent investigations. The inhibitory influence of dbc-AMP was further substantiated by decrease in soluble protein. The fungus on exposure to dbc-AMP experienced morphological differentiation from vegetative mycelial phase to sporogenous mycelial phase, and was induced to produce filiform sporidia. Use of quantitative ELISA further suggested that sporidia formation took more than 21 days in the presence of dbc-AMP. Variations of proteins during different stages ofT. indica grown in the presence and absence of dbc-AMP suggested the expression of stage-specific proteins or differential expression of proteins induced by dbc-AMP. The changes in expression of cell surface antigens as evidenced from decrease and increase binding of anti-mycelial and anti-sporidial antibodies in dbc-AMP treated culture by ELISA was further interpreted on the basis of morphological differentiation from mycelial to sporidial phase     

Fungicidal activity of Ranunculus asiaticus and other weeds against Fusarium oxysporum f.sp. lycopersici

The effects of aqueous extracts of some common weed species against Fusarium oxysporum Schlecht f.sp. lycopersici (the causal agent of tomato wilt disease) were investigated under laboratory conditions. Anagallis foemina L., Cerastium dicotomum L., Falcaria vulgaris L., Ranunculus asiaticus L., Scorpiurus mur-icatus L. and Solanum nigrum L. extracts were the most toxic to the fungus. Further studies on buttercup (Ranunculus asiaticus L.) showed that fresh shoot extract of this species prevented growth of F. oxysporum when incorporated into agar medium. Extracts of different parts of the plant inhibited fungus growth and sporulation, but the fungitoxicity decreased with incubation period with only slight changes in the toxicity of fresh shoot extract. The shoot and fresh parts extracts were more toxic than root and dried tissue extracts. Addition of 0.5 ml fresh shoot or 1 ml fresh root extract to the growing medium significantly reduced fungal colony growth, and the effect was extract concentration dependent. Fresh shoot extract of R. asiaticus added to a liquid medium significantly reduced mycelial dry weight compared with the control, and incorporation of 0.1 g dried shoot or 0.2 g dried roots in the media strongly inhibited fungus growth. Results of a pot experiment showed no harmful effects of R. asiaticus extracts on tomato growth.     

Specific inhibition of sclerotium formation by 2-mercaptoethanol and related sulfhydryl compounds in Sclerotium rolfsii.

Sclerotium formation in Sclerotium rolfsii was completely inhibited by 2-mercaptoethanol at a concentration of 2-4 mM without any adverse effect on mycelial growth. Concentrations lower than 2 mM had no effect on mycelial growth and sclerotium formation, whereas both were inhibited at concentrations higher than 4 mM. Complete inhibition of sclerotium formation with no effect on mycelial growth was also obtained by propyl mercaptan, 1-butyl mercaptan and 2-butyl mercaptan at a concentration of 0.10 mM. Sclerotium formation was also inhibited by benzyl mercaptan and thioglycolic acid at 0.15 mM and 2-4 mM concentration respectively, whereas it was only partially inhibited by L-cysteine and glutathione at 20 mM. Mycelium grown for 21 days in nutrient medium supplemented with mercaptoethanol at a concentration of 3 mM, when transferred into fresh medium without the chemical, grew normally and produced abundant mature sclerotia. Mercaptoethanol inhibited the initiation as well as the further development of young, unpigmented sclerotia. The mechanism of sclerotium formation was arrested completely when mercaptoethanol was added to the growth medium at any time between inoculation and the appearance of sclerotia of the "development" stage. It is suggested that the specific inhibitory action of mercaptoethanol could be used to study the mechanism of sclerotium formation     

Mycelial growth characteristics in a split-plate culture of four strains of the genus <Emphasis Type="Italic">Suillus</Emphasis>

A split-plate method with two media in different concentrations in each compartment was applied for the mycelial growth of four strains of Suillus luteus, S. grevillei, S. granulatus, and S. bovinus. As the glucose concentration in the A-side (the side containing higher concentrations of glucose) increased, the mycelial growth in both A- and B-sides (the side containing lower concentrations of glucose) increased. The mycelial density in both sides and B/A ratio (the ratio of the mycelial growth in the B-side to that in the A-side) also increased, and the colony morphology changed. In both A- and B-sides, the colony area reached maximum at 10g/l glucose in the A-side in most cases and at 33.3g/l in several cases. The results indicated nutrients are translocated from mycelia in the A-side to those in the B-side. High concentration of phosphate or fructose + glucose in the A-side induced better mycelial growth in the B-side. Addition of high concentrations of phosphate to one side enhanced mycelial growth in the other side. Low-temperature incubation promoted the growth in the B-side. Our split-plate culture method will be useful for qualitative study of translocation in ectomycorrhizal fungi.     

Characterization of some culture factors affecting oxalate degradation by the mycoparasite Coniothyrium minitans

Aims:  To find possible approaches to utilize the mechanism of oxalate degradation by Coniothyrium minitans (Cm) in controlling the plant pathogen Sclerotinia sclerotiorum (Ss).
Methods and Results:  Differences in oxalate degradation by different Cm strains and effects of the initial oxalate concentration, ambient pH and nutrient factors on mycelial growth and oxalate degradation by Cm were studied in shaken cultures. Results showed that two wild-type Cm strains, Chy-1 and ZS-1, did not differ in oxalate degradation in modified potato dextrose broth (mPDB) amended with oxalic acid (OA). Cm could grow in mPDB amended with sodium oxalate (SO-mPDB) at pH 6·5 or with ammonium oxalate (AO-PDB) at pH 6·2, but oxalate degradation was very low; oxalate degradation was greatly enhanced in SO- or AO-mPDB with pH being lowered to 2·8–2·9. Similarly, oxalate degradation was higher than 90% in OA-amended mPDB at pH 4·4 but was reduced to be <22% at pH 7·0. Five carbon sources and three nitrogen sources investigated and nutrients from mycelia and sclerotia of Ss were favorable for the growth of Cm and OA degradation by Cm.
Conclusions:  Cm can degrade oxalate under acidic pH. Exudates from mycelia or sclerotia of Ss may serve as nutrients for Cm mycelial growth and degradation of oxalate secreted by Ss.
Significance and Impact of the Study:  The finding of oxalate degradation laid a foundation for mining-related genes in Cm for engineering plant resistance against Ss. Elucidation of the importance of acidic pH and nutrients from Ss in oxalate degradation by Cm will help to understand the interaction between Cm and Ss.     

Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media

Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41 g/l) and EPS production (0.55 g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH2PO4, 0.1% (w/v) MgSO4 .7H2O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36 g/l).     

Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment

The snow mold fungus, Sclerotinia borealis, shows optimal growth at 4°C on potato dextrose agar (PDA) and can grow even at subzero temperature. Its mycelial growth was improved on frozen PDA at −1°C and on PDA containing potassium chloride (KCl) (water potential, −4.27 to −0.85 MPa) or d(−) sorbitol (−3.48 to −0.92 MPa). Its optimal growth temperature shifted from 4 to 10°C on PDA amended with KCl or sorbitol, indicating that inherent optimal growth occurs at high temperatures. These results suggest that S. borealis uses concentrated nutrients in the frozen environment and that such physiologic characteristics are critical for the fungus to prevail at subzero temperatures.     

Translocation induced outgrowth of fungi in nutrient-free environments

The study of alternatives to chemical methods of nematode control in agriculture has received significant recent interest. One such method is biological control using nematode trapping fungi such as Arthrobotrys superba. To understand how these fungi can be implemented as effective nematicides, it is essential to study their outgrowth into soil from localized nutrient resources. In this paper, we use a mathematical model to investigate the outgrowth of fungi into an environment essentially without available nutrients capable of supporting net growth. By comparing model solutions with experimental results, we show that in such circumstances, continual mycelial expansion can only be obtained if internal metabolites are actively translocated through the mycelium. Predictions are made concerning the maximal extension possible from a given quantity of nutrients and a testable functional relationship between the two is derived. Using this modelling technique we are able to map not only biomass extent but also biomass distribution at each stage. The type of environmental heterogeneity investigated here is encountered by many species of fungi in nature and is of relevance for the introduction of specific fungi into soil for biological control or bioremediation purposes.     

Direct quantification of in vitro cell growth through image analysis

Summary Objective, accurate, non-intrusive measurement of in vitro cell growth was realized through microcomputerized video image analysis. Recently-released video and digitizing hardware and software were incorporated into an analytical system which accurately quantified visual differences between cultures on a cell number or fresh mass basis. Sequential measurements during culture incubation further detected and quantified subtle changes in colony area and density resulting from growth. Each measurement was acquired rapidly, without encroaching on the in vitro environment, so cell growth was undisturbed. Custom software routines coordinated the quantification of this detailed record into precise cumulative growth curves.     

Dimorphic and yeast-like mutants of the genusCephalosporium Cda

A series of mutants, in which the mycelial type of growth gradually changes to the dimorphic and permanent yeast-like forms, were isolated from cultures ofCephalosporium sp. subjected to UV radiation. The intermediate stage between the mycelial and dimorphic strains (mutants 2/29 and 2/R) is characterized by the absence of aerial hyphae, ability to form conidiophores inside agar and by polymorphism of conidia. The Y-M transformation of two dimorphic mutants obtained from the 2/R mutant depends on temperature. Another mutant isolated from the 2/29 strain was found to form the mycelial phase only when osmolarity of the medium increased. At 22°C the transformation of all three dimorphic strains was influenced by the carbon source: the Y phase predominated in glucose-containing media, the M phase predominated in media with amino acids or citrate serving as carbon sources. Another mutant (2/7R) was found to grow permanently in the Y phase and was not influenced by temperature, osmolarity of the medium and by the carbon source. It is assumed that the dimorphism of the mutants is caused by a conformational mutation inhibiting the apical growth. This mutation can be phenotypically reversed by some factors of the environment.     

A preliminary study on the effects of salicylic and jasmonic acids on Ganoderma boninense growth,mycelial hydrophobicity,and media pH under in vitro assays

Potato dextrose agar amended with salicylic acid (SA) suppressed growth of all Ganoderma boninense isolates. However, mycelial growth inhibitory effects varied with times, SA concentrations and G. boninense isolates. At 150 ppm of SA or higher, growth suppression diminished with time and mycelial growth recovery was observed. In contrast, jasmonic acid (JA) unexpectedly improved growth of G. boninense. At 21 day-after-inoculation, pH changes and hydrophobicity for the melanised mycelia were lower compared to non-melanised mycelia. The degree of inhibitory or promotional effects on the growth of G. boninense and the level of G. boninense mycelial hydrophobicity by SA and JA were isolate-dependent.     

Changing medium and passaging cell lines

Cell lines are widely used in biomedical research. This protocol describes the methods used routinely to change the medium and passage the cells. Medium changes keep the cells healthy by providing fresh nutrients, while cell passage or splitting is required to maintain cells in exponential growth. Despite the simplicity of the methods used, each cell line has idiosyncracies. Whether working with one or several cell lines, there is no substitute for knowledge of their needs, including the range of phenotypes and growth patterns under different physical and nutrient conditions. Given the necessary care and attention, most cell lines are easy to maintain and grow.     

The role of nutrient availability in regulating root architecture

The ability of plants to respond appropriately to nutrient availability is of fundamental importance for their adaptation to the environment. Nutrients such as nitrate, phosphate, sulfate and iron act as signals that can be perceived. These signals trigger molecular mechanisms that modify cell division and cell differentiation processes within the root and have a profound impact on root system architecture. Important developmental processes, such as root-hair formation, primary root growth and lateral root formation, are particularly sensitive to changes in the internal and external concentration of nutrients. The responses of root architecture to nutrients can be modified by plant growth regulators, such as auxins, cytokinins and ethylene, suggesting that the nutritional control of root development may be mediated by changes in hormone synthesis, transport or sensitivity. Recent information points to the existence of nutrient-specific signal transduction pathways that interpret the external and internal concentrations of nutrients to modify root development. Progress in this field has led to the cloning of regulatory genes that play pivotal roles in nutrient-induced changes to root development.     

Synthesis of neomycin by washed mycelium ofStreptomyces fradiae and some physiological considerations

Studies on the effects of different carbon sources on neomycin formation by washed cells ofStreptomyces fradiae 3535 indicate that they do not stimulate the antibiotic synthesis. The higher titer of neomycin in mineral salts medium is due to the fresh synthesis of neomycin and not merely due to release from the mycelium. Glucosamine andN-acetylglucosamine are stimulatory to neomycin production. The neomycin activity of the broth and the alkaline phosphatase level of the mycelium decrease on the addition of glucose to the medium. The metabolism of neomycin and neomycin phosphate is stimulated in the presence of glucose. Studies on changes in mycelial constituents during neomycin production show that during lysis there is loss of amino acids from the cell while the amino sugar and sugar content remain unaffected. In the medium where cells are resistant to lysis, mycelial total amino acid, amino sugar and sugar increase gradually and the growth phase is prolonged upto day 7 of fermentation.     

STUDIES OF THE GROWTH OF PENICILLIUM CHRYSOGENUM IN CONTINUOUS FLOW CULTURE WITH REFERENCE TO PENICILLIN PRODUCTION

SUMMARY: A filamentous mould was cultured by the continuous flow method in which medium is supplied at a constant rate and the culture volume is kept constant. Flow rates up to 0·1 culture volumes/hr were used. The mycelial dry weight concentration and the yield of mycelium/g of carbon source used were equal to or slightly greater than the maximum obtained in batch culture. With glucose concentrations up to 80 g/1. at a flow rate of 0·05 culture volumes/hr, about 45% of the substrate carbon was converted into mycelial carbon and the remainder oxidized to CO₂.
With unlimited amounts of all nutrients available growth of the mould followed the exponential law, as does bacterial growth, and therefore the mould had a constant doubling time.
The oxygen demand of the mould as function of growth rate was determined.
Conditions were found under which the rate of penicillin production/g of mycelium remained at its maximum value for 1000 hr.     

Lipid synthesis during reinitiation of growth from stationary phase cultures of Candida albicans

Lipid synthesis has been studied in the dimorphic fungus Candida albicans. ¹⁴C-acetate incorporation into lipid material was used to measure new lipid synthesis in two cultures in which either yeast or mycelial growth was initiated from stationary phase yeast cells. When resuspended in fresh medium at 37 °C, cells resume growth and change morphology while at 30 °C cells resume budding growth. When resuspended at the appropriate temperature, both yeast and germ tube cultures immediately incorporated ¹⁴C-acetate into lipid material. The labeled lipid was more or less evenly divided between neutral and phospholipid. Phosphatidyl choline was the major phospholipid fraction and along with phosphatidyl ethanolamine accounted for 60–65 % of the total phospholipid. Lipid synthesis during growth initiation of either morphology showed a similar pattern, with no significant differences observed in neutral or phospholipid or phospholipid components between yeast and mycelial forms.     

Possible involvement of pleiomorphic vacuolar networks in nutrient recycling in filamentous fungi

Morphological analyses of vacuoles in filamentous fungi in the past decade have led to the remarkable finding that they are highly pleiomorphic organelles. Among them, tubular vacuoles have been implicated in nutrient transport between hyphal tips and the host plant surface in mycorrhizal fungi. However, a series of works suggested the presence of tubular vacuoles in other fungi that are not mycorrhizal, including Aspergillus oryzae, hinting at more general roles of the tubular vacuoles. Recently, we made two key observations by using the fusion protein of enhanced green fluorescent protein (EGFP) with a putative vacuolar t-SNARE in A. oryzae; tubular vacuoles formed more extensively in hyphae that were not in contact with nutrients, and vacuoles that were interconnected by tubules in the mature mycelial region displayed traces of microautophagy-mediated degradation of cytoplasm. The aim of this addendum is to discuss the possible involvement of vacuoles in degrading, transporting, and recycling nutrients from the mature mycelial region to hyphal tips, to support the continuous tip growth.     

Differences in processing dynamics of fresh and dried leaf litter in a stream ecosystem

SUMMARY. 1. Although the bulk of litter input to stream ecosystems is in the form of fresh leaves, current understanding of organic matter processing is largely founded on experimental studies made with pre-dried leaves. This paradox points to the critical need for evaluating to what extent those experiments with dried leaves reflect natural litter decomposition.
2. The mass loss rates, patterns of mass loss, and chemical changes during processing of fresh leaf litter were compared with air-dried leaf litter in a stream ecosystem.
3. Although overall mass loss rates were similar between treatments ( k = 0.0213 day⁻¹ and 0.0206 day⁻¹), fresh leaves lost mass at a constant rate, whereas the decay of dried leaves proceeded in two distinct phases. Soluble organic carbon, phosphorus, and potassium were rapidly leached from dried litter, but were largely retained in fresh material for more than a week. Kinetics of concentrations of cellulose and changes in amounts of lignin remaining per leaf pack revealed further differences in decomposition dynamics between treatments, apparently related, either directly or indirectly, to differences in leaching behaviour.
4. Dynamics of nitrogen and protein contents were similar between treatments, indicating that microbial colonization was not greatly delayed on fresh leaves.
5. It is concluded that the retention of labile carbon and nutrients in fresh leaf litter facilitates their utilization by leaf-associated micro-organisms and invertebrates, resulting in an increased importance of biotic processes relative to physical processes such as leaching.
6. At the ecosystem level, retention of carbon and nutrients in streams would be increased, allowing greater overall productivity. Conversely, the availability of labile organic carbon would be reduced in compartments such as the epilithon, fine sediments, and the water column.