Mycelial Production Research Articles

Importance of Stream Microfungi in Controlling Breakdown Rates of Leaf Litter

Breakdown of seven leaf species covering a broad range of litter qualities (lignin: 7—31% of leaf dry mass; tannin: 0.0—6.7%; nitrogen: 0.5—2.6%; phosphorus: 0.017—0.094%) and dynamics of fungal biomass and reproductive activity were studied in a softwater mountain stream. Litter breakdown proceeded at exponential rates k ranging from 0.0042 d—1 (evergreen oak) to 0.0515 d—1 (ash). Fungal colonization of litter was generally rapid, with the fungus—specific indicator molecule ergosterol increasing from initially negligible concentrations to 375—859 mg/g of detrital mass. Using species—specific factors relating ergosterol concentrations to mycelial dry mass, maximum fungal biomass associated with litter was estimated as 61—155 mg/g of total system mass. Minimum estimates of net mycelial production during active growth varied between 0.3 and 3.8 mg°g—1°d—1, and maximum sporulation rates of aquatic hyphomycetes ranged from 760 to 7500 conidia°mg—1°d—1. Initially, reproductive activity was largely synchronized with increases in ergosterol concentrations, but it declined dramatically after peak sporulation rates were reached, whereas ergosterol concentrations levelled off or decreased at considerably slower rates. Periods of highest fungal productivity were thus limited to an initial breakdown stage of °2—8 wk. Strong correlations were found between the exponential breakdown coefficient and each of three parameters reflecting fungal activity in leaf litter, that is, maximum ergosterol concentration (P = 0.002, r = 0.96), net mycelial production (P = 0.02, r = 0.92), and sporulation rate (P < 0.001, r = 0.99). The initial lignin content of leaves was also significantly correlated with the rate constant k (P = 0.02, r = —0.83), suggesting that lignin was the primary factor determining the litter quality and thus breakdown rate. The correlation was even stronger when data were logarithmically transformed (P < 0.01, r = 0.95). Tannin concentration was significantly correlated with k only when two high—lignin species were excluded from the analysis (P = 0.19, r = 0.56 compared with P = 0.05, r = —0.88), while initial concentrations of phosphorus (P = 0.17, r = 0.58) and particularly nitrogen (P = 0.82, r = 0.06) were poor predictors of litter decomposability. These results suggest that the initial lignin content of leaves controlled litter breakdown rate through a kinetic limitation of carbon sources for saprotrophic microfungi. The decomposer activity of these organisms, in turn, would then have governed breakdown rates. In doing this, fungi produced substantial amounts of both mycelial and conidial biomass that was potentially available to higher trophic levels of the food web.

Production, formulation and application of the entomopathogenic fungusBeauveria bassianafor insect control: current status

This review summarizes the progress and achievements made in the last decade in mass production formulation and application technology of the entomopathogenic fungus Beauveria bassiana. Reports published on relevant research from Belgium, Canada, China, Cuba, Czechoslovakia (former), France, Germany, Great Britain, Philippines, Poland, Switzerland, USA and USSR (former) regarding this topic have been covered. Much of the non‐English language literature, particularly that from Eastern European and Chinese sources, has not been translated and is inaccessible to most English or other western language readers. We have done this translation and through this review provide technological details about mass production of B. bassiana in China. Various aspects of B. bassiana growth, substrate use, production of mycelia, conidiospore and blastospores, process technologies associated with separation, drying and milling, formulation, storage and ‘shelf‐life’, and field efficacy are reviewed. Data are presented on: a m...

Variation in Nitrogen Utilization in Acremonium coenophialum Isolates

Acremonium coenophialum, a fungal endosymbiont in tall fescue, is responsible for the production of alkaloid toxins. Animals grazing endophyte-infected tall fescue often show toxicosis. In marginal environments, the endophyte is important for long-term survival of tall fescue. Few differences in endophyte isolates from individual tall fescue plants have been reported. To aid development of a toxicosis-free tall fescue, it is important to identify differences in endophyte isolates. This report describes variation in nitrogen utilization in a defined culture medium by endophyte isolates from Kentucky-31 tall fescue. Overall, the best nitrogen sources for dry weight production of mycelium were proline and potassium nitrate. Thirty-four isolates grown on agar-solidified defined media with single nitrogen sources showed variation in nitrogen utilization. Fifty percent of the isolates were unable to utilize two or more amino acids. Manipulation of endophyte variation could lead to development of a toxicosis-free tall fescue cultivar.

Influence of iturin A on mycelial weight and aflatoxin production byAspergillus flavus andAspergillus parasiticus in shake culture

Iturin A, a peptidolipid produced byBacillus subtilis, inhibits growth of a large number of fungi. In this study, the effects of iturin A were evaluated on nine isolates ofA. flavus and seven isolates ofA. parasiticus in liquid shake culture. The mycelial dry weight of theA. flavus isolates was not significantly influenced by iturin A, however, there was a significant reduction in mycelial dry weight for two of theA. parasiticus isolates. Aflatoxin production was significantly reduced in five of theA. flavus isolates and three of the six aflatoxigenicA. parasiticus isolates. For the other seven isolates, aflatoxin levels were either unchanged or significantly increased in the presence of iturin A. These results indicate that iturin A does not consistently reduce growth or aflatoxin production of these fungi in pure culture.

Toxicity and Pathology of Crude Broth Filtrate Produced by Hirsutella thompsonii var. thompsonii in Shake Culture

Metabolic products secreted by the fungal mycelia of Hirsutella thompsonii var. thompsonii (CBS 556.77D) in a defined culture broth in shake culture were tested for toxicity to Galleria mellonella larvae and Drosophila melanogaster adults via injection and per os application, respectively. In addition, the toxic effect of broth filtrate was observed in vitro in a cell line of Bombyx mori. Czapek-Dox broth fortified with 1% yeast extract stimulated more rapid mycelial growth and correspondingly more toxin production in time. At 25-30°C, metabolic toxin(s) was detected in broth via bioassay at about 4-5 days postinoculation when mycelial biomass reached 5 mg/ml (dry wt). At these temperatures, biological activity of the filtrate peaked at about 8-10 days when mycelial growth reached a maximum (10 mg/ml, dry wt). This suggests a positive relationship between toxic metabolite and mycelial production. After 10 days, the toxicity of the filtrate appeared to decline gradually. Pathogenicity symptoms of the metabolites developed slowly in both G. mellonella and D. melanogaster . Early signs of lethargy appeared at 4 days postinjection and cumulative mortality of G. mellonella larvae was low after 1 week; however, the percentage of mortality reached 98-100% after 14 days. At death, G. mellonella larvae displayed small dark spots on a brownish cuticle. Histopathological effects were observed in the larval midgut, malpighian tubules, hypodermis, fat body, hemocytes, muscle, and silk glands. Cellular change consisted of pycnosis of the nucleus and a reduction in cytoplasm density. Highest mortality (78.8%) to adult D. melanogaster occurred after 10 days post-treatment. A strong cytotoxic effect was detected in B. mori cells after 24 hr.

Mycelial differentiation of Morchella esculenta in pure culture

The development of mycelia derived from single or several ascospores of Morchella esculenta was studied on two nutrient media and at different temperatures. The cultures displayed distinctive types and patterns of production of sclerotia, associated with variation in the production of aerial mycelium and expression of non-self-recognition reactions.

Efficient production of mycelium of Lentinus edodes by a continuous culture and the effect of lignin on growth

In order to obtain a high yield of Lentinus edodes mycelium efficiently used as a liquid spawn for Shiitake mushroom production, a continuous culture system was investigated. The productivity of the mycelium (mg of dry weight of mycelium ml −1h −1) was 14-fold higher at a dilution rate of 0.03 h −1 than that in a batch culture. In the continuous culture, the pulpy form of the mycelium could be easily obtained either with a low dilution rate less than 0.01 h −1 or high agitation conditions of more than 400 rpm. The change in morphology to pulpy form resulted in a increase in the dry weight of the mycelium (DW). The maximum DW was 20 mg/ml at a dilution rate of 0.01 h −1. Lignin, which has been reported to accelerate the growth of L. edodes in a submerged culture, had no effect on mycelium production in the continuous culture, but promoted both the specific glucose consumption rate and maintenance energy. In addition, it was observed that lignin improved the resistance of the mycelium against shear stress. Adding 0.1% acetic acid to the feed medium and maintaining a low pH (about 4.0) in the culture allowed operation for over 70 d without any microbial contamination.

Effects of excess nitrogen and phosphorus starvation on the extramatrical mycelium of ectomycorrhizas of Pinus sylvestris L.

summaryThe effect of excess nitrogen alone, and in combination with phosphorus and magnesium starvation, on the production of extramatrical mycelium was studied in Scots pine seedlings ectomycorrhizal with Laccaria bicolor (R. Mre.) Orton, Hebeloma crustuliniforme (Bull, ex St‐Amans) Quel. and Suillus bovinus (L. ex Fr.) O. Kuntze. Seedlings were grown in a semi‐hydroponic cultivation system and the ergosterol assay was used to estimate fungal biomass. The mycelial biomass increased rapidly when N was kept low (10–20 mg l−1) and in balance with other nutrients, but no extramatrical mycelium was produced when the N concentration was raised to 200 mg l−1. The growth of the extramatrical mycelium resumed when the excess N treatment was terminated and the seedlings were returned to a low nutrient regime, Laccaria showing more complete resumption of growth than Suillus, while Hebeloma had a low production of extramatrical mycelium in all treatments. Compared with the external mycelium, the amount of fungal tissue on the mycorrhizal roots (mantle and Hartig net) was much less affected by the high N treatment. P starvation increased the production of extramatrical mycelium tenfold, with almost no difference between high and low N nutrient regimes, while Mg starvation had no effect on the fungal biomass. The high N treatment lowered P and particularly Mg concentration of the needles, regardless of the mycorrhizal status of the plant.It is suggested that nitrogen deposition from the atmosphere may damage the function of mycorrhiza even before root tip studies reveal any decline in the symbiotic state. On the other hand, moderate nitrogen fertilization of forest land unaffected by nitrogen pollution is likely to have only passing effects on mycorrhizal development.

Effects of culture conditions on amylase production by some soil fungi

Twenty-one species of soil fungi have been screened for amylolytic activity. Six species designated as active enzyme producers were used in the experiments: Aspergillus flavus, Cladosporium herbarum, Cunninghamella echinulata, Fusarium culmorum, Paecilomyces divaricata, and Penicillium notatum. The results obtained revealed that, in most cases, enzyme production and dry weight yield of mycelium were not positively correlated. An incubation period of 8 days at 27°C and an initial pH range of 4–6 were most favourable for enzyme production by all fungi studied. A 1.5% concentration of soluble starch was the most suitable substrate for all the studied fungi although rice and corn flours appeared to be preferred by A. flavus, F. culmorum, P. divaricata and P. notatum. Ammonium nitrate at 0.5% concentration provided a suitable nitrogen source for all fungi although productivity by A. flavus and P. divaricata was enhanced by the presence of casein and urea. A C: N ratio of 3:1 was the most favourable for all fungi investigated. A concentration of 10 μg/L SrCl2 favoured productivity by all fungi. Preferential utilization of nutrients caused changes in pH towards alkalinity which repressed productivity. 21 Bodenpilz-Arten wurden auf ihre amylolytische Aktivität untersucht. 6 Arten waren aktive Enzymproduzenten und wurden weiter optimiert: Aspergillus flavus, Cladosporium herbarum, Cunninghamella echinulata, Fusarium culmorum, Paecilomyces divaricata und Penicillium notatum. Die Ergebnisse offenbarten, daß in der Mehrzahl der Fälle die Enzymproduktion und der Trockengewichtsertrag des Myzels nicht positiv korrelierten. Bei allen untersuchten Pilzen war für die Enzymproduktion eine Inkubationszeit von 8 Tagen bei 27°C und ein Start-pH-Wert von 4–6 am günstigsten. Eine Konzentration von 1,5% löslicher Stärke war am geeignetsten, Reis und Korngetreidemehl wurden von A. flavus, F. culmorum, P. divaricata und P. notatum vorgezogen. Ammoniumnitrat (0,5%) ist die günstigste N-Quelle, allerdings wurde bei A. flavus und P. divaricata durch Anwesenheit von Casein und Harnstoff die Produktivität erhöht. Ein C : N-Verhältnis von 3:1 war für alle Pilze am günstigsten. Bevorzugte Verwertung von Nährsubstrat verursacht eine pH-Veränderung zur Alkalität, welche die Produktivität erniedrigt.

Inhibition of aflatoxin production inAspergillus flavus by natural coumarins and chromones

The activities of three natural coumarins, xanthotoxin and bergapten (fromAmmi majus, Umbelliferae) and psoralene (fromFicus cycomorus, Moraceae), were tested against mycelial growth and aflatoxin production of a toxigenic strain ofAspergillus flavus grown in a rice/corn steep liquor medium. Two other natural chromones, khellin and visnagin (fromAmmi visnaga) were also compared. Complete inhibition of aflatoxin release occurred with either xanthotoxin or khellin at 5 mM. The other three compounds also at 5 mM reduced aflatoxin to 12 to 16% of its original concentration. The mould growth was only slightly inhibited by all the compounds used.

Rhizopus oligosporus grown on natural rubber waste serum for production of single cell protein: a preliminary study

Maximum production of mycelium and utilization of total organic carbon byR. oligosporus grown on natural rubber waste serum was achieved at 28°C with an inoculum size of 7.5% (v/v) and grown for 144 h with an initial pH of 4.0. The maximum production of total crude protein, however, was when culture medium was inoculated with 2% (v/v) of spore suspension under the same conditions. Natural rubber waste serum may be a potential substrate for the production of single cell protein.

The effect of carbon sources on the single cell proteins and extracellular enzymes production byChrysonilia sitophila (TFB 27441 strain)

The single cell protein and extracellular enzyme production from a lignocellulolytic fungus,Chrysonilia sitophila, using different carbon sources were evaluated. The mycelial dry mass composition showed a high protein (39.2%) and low nucleic acid content (3.3%), as well as carbohydrate, fatty acid, fiber, and ash levels comparable with single cell proteins currently studied. Mycelial protein showed amino acid content similar to or higher than FAO standard requirements. The amino acid, fatty acid, and carotenoid composition, as well as mycelial mass yield and enzyme production, were dependent on the carbon source used. Glucose, saccharose, cellobiose, cellulose, microcrystal-line cellulose, lactose, and rice hull as carbon sources were studied.

In vitro production of pycnidia by Septoria tritici

The in vitro production of pycnidia bySeptoria tritici was examined on six media reported to induce the formation of fruiting bodies. Among 26 freshly isolated cultures from various parts of the world, consistent differences in growth type were found which were only partially influenced by nutritional and environmental conditions. Cultures with yeast-like growth produced hardly any pycnidia or pseudopycnidia, while cultures with intermediate or mycelial growth types produced them frequently. Incubation in continuous darkness induced intermediate to mycelial growth types rather than yeast-like growth types in some cultures, and concomitantly the production of more pycnidia. Potato-dextrose agar induced intermediate to mycelial growth types and production of (pseudo)pycnidia more often than V8 agar and wheat leaf extract agar, which had previously been reported to be especially beneficial to (pseudo) pycnidium formation byS. tritici. Isolates with a consistently yeast-like growth type, producing (virtually) no fructifications under any of the experimental conditions, were slightly stimulated to form pseudopycnidia on water agar supplemented with sterile pieces of maize, wheat or carnation leaves.

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.

Allergens as proteases: An aspergillus fumigatus proteinase directly induces human epithelial cell detachment

Allergic bronchopulmonary aspergillosis (ABPA) is characterized by pulmonary and systemic allergic and inflammatory processes triggered by fungal antigens. Airway damage is a feature of this disorder, and although Aspergillus-derived proteinases have been described, the capacity of Aspergillus, however, to directly induce damage to human epithelium has not previously been studied. We therefore cultured Aspergillus fumigatus from two patients with ABPA, extracted mycelial products by sonication and filtration, and then evaluated their capacity to induce epithelial cell (EC) desquamation from basement membrane using an in vitro model that uses intact human amniotic EC and native basement membrane. A. fumigatus extracts induced detachment of EC in a dose-dependent fashion, producing up to 34% +/- 6% detachment (p less than 0.05 compared to medium alone). Enzyme analysis of A. Fumigatus extract using synthetic substrates revealed the presence of a number of different enzymes; therefore, studies with specific proteinase inhibitors were undertaken to identify the proteinase(s) responsible for detachment. A. Fumigatus-induced desquamation was partially inhibited by phenylmethylsulfonylfluoride and substantially inhibited by glutathione and N-acetylcysteine, but not by alpha 1-antitrypsin, 1,10 phenanthroline, ethylenediaminetetraacetic acid, aprotinin, or soybean trypsin inhibitor at concentrations that inhibit other serine- or metalloproteinases. Gel filtration of the extract with a Sepharose 6B column revealed that the major epithelium-detaching activity appeared in the 20 to 35 kd fraction. Comparison with proteinase standards suggested a role for a chymotrypsin-like proteinase. Thus, A. fumigatus releases a proteinase that is directly able to induce EC detachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10 and 10 spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 x 10 to 2 x 10 spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.

Tansley Review No. 19 Environmental and endogenous controls of developmental pathways: variation and its significance in the forest pathogen, Heterobasidion annosum.

Cultural studies of the forest root pathogen Heterobasidion annosum (Fr.) Bref. have shown that the mycelium of the fungus possesses a high degree of developmental versatility, being able to grow in a variety of distinctive functional modes. Consequently the fungus can alternate between conidiogenous and meiotic reproductive pathways, rapid, sparsely branched and slow, densely branched mycelial extension, nutritive and non-nutritive modes, and'juvenile'and 'senescent'phases. This ability is probably of considerable importance in natural infection cycles and provides an interesting model for examining interrelationships between developmental regulation and host-pathogen interactions generally. It also raises important questions about the interface between environmental and endogenous controls of developmental pathways. CONTENTS Summary 245 I. Introduction 245 II. Spore germination and mycelial extension 246 III. Production of non-nutritive mycelium 249 IV. Sporophore production, senescence, somatic incompatibility and extracellular enzyme production 251 V. Discussion 254 Acknowledgements 255 References 255.

Nutritional requirements of the mycoparasitic fungus Verticillium biguttatum

Verticillium biguttatum was able to grow axenically in a synthetic liquid medium with a compound containing ammonium or amino group as nitrogen source, glucose as carbon source and biotin as growth factor. Among various carbon and nitrogen compounds tested, highest mycelial production was achieved with mannitol and with two ammonium salts and glutamine; sporulation reached highest values with galactose and glutamine. Highest yields of mycelium and conidia were obtained at pH 4.3 and 5.1, respectively. Although neutral and alkaline conditions were growth-limiting in the synthetic medium some growth ofV.biguttatum occurred on solid media at pH 7.0 and on sclerotia ofRhizoctonia solani in natural soil at pH 7.2–7.3.

Mass Production of the Endoparasitic Fungi Drechmeria Coniospora, Ver Ticillium Balanoides and Harposporium Anguillulae in Liquid Culture

Mycclial growth and rate of sporulation of Drechmeria coniospora, Verticillium balanoides and Harposporium anguillulae were tested in different concentrations on liquid corn meal (CM) and on potato dextrose (PD) media. Mycelial growth of all three fungi was greatest in PD, whereas conidial production was highest in CM. Spore productivity increased in CM and decreased in PD with increasing nutrient levels. Mycelial production was greatest at high nutrient concentrations of both media. Of the three fungi, the highest levels of growth and sporulation were achieved by V. balanoides. Conidia of the endoparasitic fungi produced in liquid culture retained their parasitic capabilities.

Lipolytic activities of two strains of Aspergillus aculeatus associated with the spoilage of two food sources

AbstractThe production of extracellular lipolytic enzymes by 2 strains of Aspergillus aculearus associated with the deterioration of peanuts and a marketed margarine in Nigeria and the effects of temperature and pH on the activities of the enzymes were investigated. Lipolytic enzymes were detected within 2 days of incubation at 35 °C in 4 out of 6 natural oils and in 3 out of 4 synthetic glycerides used irrespective of strain type. The lipolytic enzymes of both strains hydrolysed both natural oils and synthetic glycerides to free fatty acids. There was correlation between the mycelium produced to the quantity of free fatty acids produced in medium within the first 8 days of incubation. There was a general increase in mycelium production with increase in incubation period. The enzyme activities were at peak at 30‐35 °C and at pH 6‐7.