Production of Destruxins from Metarhizium spp. Fungi in Artificial Medium and in Endophytically Colonized Cowpea Plants

Destruxins (DTXs) are cyclic depsipeptides produced by many Metarhizium isolates that have long been assumed to contribute to virulence of these entomopathogenic fungi. We evaluated the virulence of 20 Metarhizium isolates against insect larvae and measured the concentration of DTXs A, B, and E produced by these same isolates in submerged (shaken) cultures. Eight of the isolates (ARSEF 324, 724, 760, 1448, 1882, 1883, 3479, and 3918) did not produce DTXs A, B, or E during the five days of submerged culture. DTXs were first detected in culture medium at 2–3 days in submerged culture. Galleria mellonella and Tenebrio molitor showed considerable variation in their susceptibility to the Metarhizium isolates. The concentration of DTXs produced in vitro did not correlate with percent or speed of insect kill. We established endophytic associations of M. robertsii and M. acridum isolates in Vigna unguiculata (cowpeas) and Cucumis sativus (cucumber) plants. DTXs were detected in cowpeas colonized by M. robertsii ARSEF 2575 12 days after fungal inoculation, but DTXs were not detected in cucumber. This is the first instance of DTXs detected in plants endophytically colonized by M. robertsii. This finding has implications for new approaches to fungus-based biological control of pest arthropods.     

Localization of β-1,3-glucanase in Trichoderma harzianum

Studies on the constitutive β-1,3-glucanase were conducted in submerged as well as in the stationary culture conditions, in the presence and in the absence of lactose and glucose as main carbon sources. In the absence of lactose or glucose, expression of β-1,3-glucanase was observed at 96?h in extracellular, periplasmic, cell wall bound and internal fractions during submerged fermentation. In shake flask culture, enzyme was found in all subcellular fractions using optimal glucose concentration. When Trichoderma harzianum was grown on media containing 55?kg lactose/m³ in submerged culture, activity was found in extracellular, cell wall bound and in the periplasmic fractions. The relative distribution of the enzyme in the cell is independent of the nature of the carbon source and its concentration.     

Degradation of Chrysanthemum (Dendranthema grandiflora) wastes by Pleurotus ostreatus for the production of reducing sugars

In Colombia, a great amount of waste is generated during the cut-off and harvest stages in flowers culture. This study examines the possibility of degrading Chrysanthemum wastes by using Pleurotus ostreatus, Trametes versicolor, and Phanerochaete chrysosporium cultures; this has not been studied previously. The initial effect of fungi on the degradation of Chrysanthemum wastes were studied individually and in co-cultures. The highest degradation was by P. ostreatus. After that, the influence of pH and waste, copper, and manganese concentrations on reducing sugars concentration were determined in a submerged culture in 100 mL Erlenmeyer flasks. There was a significant effect of manganese and waste concentrations on sugar concentration, while the effect of copper concentration and pH were not significant. Following, the process was carried out in a 1.5 L reactor at the optimal values of the variables studied in Erlenmeyer flask but varying air injection from 0 to 2 vvm. The highest concentration of sugars was 21.2 g/L with 78% of glucose content at 6.3% w/v of waste, 7.5 mM of Mn and Cu and 2 vvm of air injection. Finally, laccase, Manganese peroxidase, endo-1,4-β-glucanase, exo-1,4-β-glucanase and 1,4-β-glucosidase were detected in the extract obtained under these conditions. The highest activities were obtained for laccase (4,694 U/L) and 1,4-β-glucosidase (9,513 U/L).     

Ricerche Sulla Cultura Sommersa di Cellule Singole di Piante Superiori

Abstract

Research on submerged culture of single cells of higher plants. — The author describes a method which allows to obtain submerged cultures of single cells of Phaseolus vulgaris and Nicotiana tabacum. The medium composition in macroelements in the culture on agar appears to effect to a great extent the ability of tissues to dissociate into single cells in the subsequent liquid culture. In this respect Heller's solution results to be more suitable than Gautheret's and Hildebrandt and Ri-ker's.

Cells are grown at 24 [ddot]C in 300 ml flasks containing 60 ml of broth on a rotary shaker at 220 rpm.

To prevent contaminations some antibacterial agents were added to cultures of Phaseolus vulgaris. Among these Penicillin and Neomycin were not tossic at 20 and 5 ppm concentrations respectively.

The presence of septa, which are observed also in largely vacuolate cells, seems to confirm the ability of single cells to divide.

The optimum 2,4-D concentration for growth decreases from 6 × 10^-8 to 6 × 10^-8 during successive liquid cultures, each of them being inoculated with on amount of the previous one. This fact, showing the adaptation of liquid cultures to decreasing concentrations of the growth hormone, is in agreement with previous observations in solid cultures by several authors.     

Effects of Neem Leaf Volatiles on Submerged Cultures of Aflatoxigenic Aspergillus parasiticus

Microbe-free compressed air was passed continuously for a 3-day test period through an enclosed system containing fresh neem leaves; the resultant emitted volatiles were passed over the surface of submerged liquid cultures of a wild-type aflatoxigenic isolate of Aspergillus parasiticus. Aflatoxin determinations for the fungal culture that received neem-derived volatiles, after a 3-day incubation period, resulted in a 90% overall reduction in aflatoxin production and a 51% reduction in fungal biomass when compared with cultures that did not receive neem volatiles. In a separate experiment but in a similarly enclosed system, volatiles from fresh neem leaves were collected on a small Tenax column and were thermally desorbed and cryogenically focused on a capillary gas chromatography column. The neem volatiles were subsequently separated and identified by gas chromatography-mass spectrometry. Sixty-eight compounds were identified by comparison of retention times and mass spectra with either authentic compounds or spectra from a computer-assisted library database of mass spectra. It was found that 10% of the total headspace volatiles were composed of C₃ to C₉ alkenals, which are toxic to aflatoxigenic Aspergillus spp., which could explain the bioactivity that resulted in reduced biomass in the neem-treated cultures.     

Utilization of agro-industrial orange peel and sugar beet pulp wastes for fungal endo- polygalacturonase production

The pectinase enzymes are involved in several industrial applications, and industrial waste is one of the largest environmental pollutants, so this study aims to Endo-polygalacturonase (endo-PG) producing using Aspergillus niger AUMC 4156, Penicillium oxalicum AUMC 4153 and P. variotii AUMC 4149 by using some agro-industrial wastes (dried orange peel and sugar beet pulp) as a sole raw carbon source for degradation these waste in the process of urban wastes disposal. The fermentation process was carried out as a submerged culture technique under both shaken and static culture conditions. A. niger AUMC 4156 was the most promising producer of endo-PG under static conditions while P. oxalicum AUMC 4153 was the highest producer of endo-PG under shaken conditions. Sugar beet pulp proved to be the most preferable to orange peel as the only source of carbon in both shaken and static cultures. The medium that encompassing orange peel as a single carbon source afforded the highest protein content with all tested fungal strains in stirred and static cultures in comparison with sugar beet pulp. The highest activity of endo-polygalacuronase that produced using A. niger AUMC 4156 and P. oxalicum AUMC 4153 was achieved by using sugar beet pulp at 3% concentration under static cultures, meanwhile maximal enzyme activity produced by both fungal strains required 2% sugar beet pulp under shaken cultures. Sugar beet pulp showed promised potential as a good inducer for endo-polygalacturoase production, and enzymes production depended on fungal strains, culture medium, and submerged fermentation conditions.     

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

Effect of cultivation conditions on invertase production by hyperproducing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolates

Invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) finds major uses in confectionery and in the production of invert syrup. In the present study, we report on invertase production by wild cultures of Saccharomyces cerevisiae. The yeast strains were isolated from dates available in a local market. Five hyperproducing yeast strains (>100- fold higher invertase activity) were kinetically analysed for invertase production. Saccharomyces cerevisiae strain GCA-II was found to be a better invertase-yielding strain than all the other isolates. The values of Q_p and Y_p/s for GCA-II were economical as compared to other Saccharomyces cultures. The effect of sucrose concentration, rate of invertase synthesis, initial pH of fermentation medium and different organic nitrogen sources on the production of invertase under submerged culture conditions was investigated. Optimum concentrations of sucrose, urea and pH were 3, 0.2 (w/v), and 6 respectively. The increase in the enzyme yield obtained after optimization of the cultural conditions was 47.7%.     

Determination of the parameters for producing a biobinder from wood: A mathematical modeling of the transformation of lignocellulose substrate by the fungus Panus tigrinus

A biochemical scheme for the transformation of wood lignocellulose during enzymatic hydrolysis of polysaccharides and lignin destruction in reactions involving free radicals was developed, and a corresponding mathematical model was constructed. Processing (fermentation) of wood particles by the fungus Panus tigrinus in a submerged culture for producing a biobinder of wood composites—woodchip boards and fiberboards—is considered. The mathematical model was used to study the technological parameters that influence the production of enzymes and fungal biomass and the level of free radical accumulation in the substrate, i.e., the factors determining the production of the biobinder. The optimal values of these parameters were determined, namely: the specific surface of wood particles, amounting to 2000 cm²/g; processing time of 56 h; and an initial concentration of 3.0 g/l of fungal biomass in the submerged culture.     

Endospore formation by <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> in submerged culture

The ability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycle on synthetic medium CP1 with glucose under phosphate limitation and (2) in autolysing cell suspensions of high density obtained by tenfold concentration in phosphate buffer (pH 7.2) with 0.2% CaCl₂ of stationary-phase cells grown in synthetic medium. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to retain the main species-specific features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in surface cultures.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 204–214.Original Russian Text Copyright © 2005 by Filippova, Gorbatyuk, Poglazova, Soina, Kuznetsov, El-Registan.     

Enhancement of exopolysaccharide production from Ganoderma lucidum using a novel submerged volatile co-culture system

Based on the impact of volatile organic compounds (VOCs) on secondary metabolite pathways, a novel submerged volatile co-culture system was constructed, and the effects of thirteen fungal and bacterial VOCs were investigated on Ganoderma lucidum exopolysaccharides production. The results demonstrated at least a 2.2-fold increase in exopolysaccharide (EPS) specific production yield in 6 days submerged volatile co-culture of G. lucidum with Pleurotus ostreatus. Therefore, P. ostreatus was selected as a variable culture, and the effects of agitation speed, inoculum size, initial pH, and co-culture volume on EPSs production were investigated using a Taguchi L₉ orthogonal array. Finally, the highest concentration of EPSs (3.35 ± 0.22 g L^?1) was obtained under optimized conditions; initial pH 5.0, inoculum size 10%, 150 rpm, and 3:1 volume ratio of variable culture to main culture.     

The relationship between calcification and photosynthesis in the coccolithophorid Pleurochrysis carterae

Coccolithophorids are one of the dominant groups of marine phytoplankton. They are found in large numbers throughout the surface euphotic zone of the ocean, and are able to form large-scale blooms that persist for long periods of time. Coccolithophorid cells are covered by species-specific calcium carbonate crystals of various structures. In the process of calcification in coccolithophorids, Ca²⁺ is absorbed into cells from the culture medium, and a coccolith unit is formed inside the cell. Then, the coccolith unit extrudes to the cell surface where it is constructed into crystal layers. The formation of these crystals is regulated by cellular metabolism under different environmental conditions. The carbon biogeochemical cycle in the coccolithophorids involves both photosynthetic and calcification processes, which not only play an important role in population dynamics, but also in the global carbon cycle and climate change. However, one important question remains, namely, whether the relationship between photosynthesis and calcification is species-dependent. Previous studies have yielded controversial results, even in the same species. In this paper, we selected Pleurochrysis carterae, a coccolithophore species that frequently blooms in coastal areas, to study the relationship between calcification and photosynthesis. First, we studied population growth in a batch culture over several days. For batch cultures, P. carterae was inoculated into a 10 L bioreactor at an initial cell density of approximately 5 × 10⁴ cells mL^-1. The culture conditions were optimal for cell growth. Dissolved oxygen (DO) was detected during all the culture period, and the rate of photosynthetic oxygen evolution was calculated according the DO changes during the 12-h illumination period. Algal samples (10 mL) were collected during the population growth phases. The calcium carbonate content on the cell surface was determined each day by chemical titration. Next, we studied the relationship between photosynthesis and calcification at the cellular level by observing patterns of recalcification during a 12-h period. In this study, non-calcified cells were obtained by decalcifying calcified cells collected during the exponential growth period in MES-NaOH buffer solution (pH 5.5). The non-calcified cells were inoculated into culture media containing different concentrations of Ca²⁺ (0, 5, 20, 40, 50, or 100 mg L^-1). The rate of recalcification was determined by microscopic analyses in which the number of recalcified cells per 100 cells was counted at 0, 3, 6, 9, and 12 h of culture. Ca²⁺ absorbed into the cell was detected by measuring the fluorescence intensity of Fluo-3/AM labeled Ca²⁺. The rate of photosynthetic oxygen evolution in the non-calcified cell cultures was detected by measuring the changes in dissolved oxygen during the 12-h illumination period. The results showed that during the population growth period, the rate of photosynthetic oxygen evolution was inversely related to the calcium carbonate content per cell. When the amount of calcium carbonate on the cell surface increased, the relative photosynthetic ability (the rate of photosynthetic oxygen evolution) decreased, and vice versa. Both recalcification rates and photosynthetic oxygen evolution were affected by the extracellular calcium concentration. Non-calcified cells showed different recalcification abilities at different extracellular Ca²⁺ concentrations. The recalcification rate of non-calcified cells was positively correlated with the extracellular calcium concentration when [Ca²⁺] in the medium ranged from 0 to 100 mg L^-1. However, photosynthetic oxygen evolution was suppressed at higher cell calcification rates, especially when extracellular [Ca²⁺] was 50–100 mg L^-1. Our analyses of the population growth process and the cell recalcification process confirmed that photosynthesis is inversely related to calcification in P. carterae.     

Detection of soluble immune complexes by their binding to Fc receptors on mastocytoma cells

Adenosine kinase activity in in vitro human peripheral blood monocyte and human pulmonary alveolar macrophage cultures undergoes significant increases, 3- to 10-fold, in both total and specific activity during 14 days culture. Increased activity in monocyte cultures was not detected during the first 3 days of culture. Adenosine kinase activity in both mononuclear phagocyte cell cultures had a pH optimum at 6.0 and activity was dependent on the concentration of ATP and magnesium; 5 mM ATP and 2.5 mM MgCl were optimal. Increased concentrations of ATP or magnesium were inhibitory. Both dATP and GTP served as phosphate donors in the absence of ATP; in contrast, pyrimidine triphosphates were poor donors. Enzyme activity was inhibited by 1 μM p-chloromercuribenzoate and substrate inhibition by excess adenosine was observed in 2-week pulmonary alveolar macrophage cultures but not in freshly isolated cells. The role of increased adenosine kinase activity in in vitro monocyte-macrophage differentiation is considered.     

Conversion of cassava flour to fuel ethanol by sequential solid state and submerged cultures

A process for conversion of cassava flour to ethanol was developed. This involved direct inoculation of Aspergillus awamori spores into a cassava flour paste and incubation for some period during which hydrolytic enzymes are produced (solid state culture or koji production) and subsequent addition of water and yeast cells, during which there is simultaneous hydrolysis and ethanol production (submerged culture). When cassava flour alone was used for the solid state phase, the paste was very sticky, making mixing and aeration difficult. However, addition of rice bran improved the texture and enzyme production. The optima rice bran concentration, spore inoculum concentration, and duration of solid state culture before submerged culture were 20%, 6.16 × 10⁶ spores/100 g, and 2 days, respectively. Under these optimum conditions, a high ethanol concentration of 120 g/L and ethanol yield of 0.309 g-ethanol/g-cassava flour were obtained. This ethanol yield corresponds to 0.44 g-ethanol/g-cassava starch.     

Improved cordycepin production in a liquid surface culture of <Emphasis Type="Italic">Cordyceps militaris</Emphasis> isolated from wild strain

A Cordyceps militaris NBRC 10352-3 strain that was isolated from C. militaris NBRC10352 produced 68 mg of cordycepin from 100 mL of medium, which was the highest level of cordycepin among 60 isolates from three C. militaris (NBRC 9787, 100741 and 103752) strains. Interestingly, a liquid surface culture of C. militaris NBRC 103752-3 produced 2-fold cordycepin to that in a submerged culture. Cordycepin production was significantly affected by specific surface area (SSA) in the liquid surface culture, and 120.9 mg of cordycepin was produced on SSA of 1.57/cm (from 50 mL medium). The addition of glycine and adenine as an additive to its culture medium was optimized by an experimental design. When 6.75 g/L of adenine was added to the culture, 312.2 mg of cordycepin (apparent concentration: 6.2 g/L) was produced from 50 mL medium, improving the cordycepin production by 4.6-fold. In this study, the production and productivity of cordycepin were significantly improved in C. militaris wild type by a single cell colony isolation and additives without adopting any mutational technologies. This C. militaris NBRC 10352-3 strain can be used as a new cordycepin-hyperproducing one, instead of a cordycepin-hyperproducing mutant.     

Comparative study of cultures of four species of the genusOudemansiella

Thirty stationary and submerged cultures of species of the genusOudemansiella Speg.,viz. two narrow-region species (O. brunneomarginata, O. canarii) and two primarily wideregion species (O.mucida, O. radicata) were compared. Stationary cultures have identical macroscopic and microscopic features (they produce crust, teleomorph, large thick-walled basidiospores with an apiculum and an uneven surface, dikaryotic hyphae have clamp connections). “Coils” were detected in the mycelium of two species by SEM. Submerged cultures produce antibacterial and particularly antifungal antibiotics, although with various intensity.O. brunneomarginata was first studied in pure culture and its microstructures were first studied by SEM. It could be demonstrated that this species is related with other representatives of the genusOudemansiella. In the present taxonomy of basidiomycetes it is advisable to extend and deepen data concerning a single genusOudemansiella with a series of interspecies taxons, without impairing “borders” of the genus.     

Conidiation of Hirsutella thompsonii var. synnematosa in submerged culture

Fourteen monosporal isolates of Hirsutella thompsonii grew vegetatively in various liquid media producing typical phialidic-like conidiophores. Hirsutella thompsonii var. synnematosa from Ivory Coast (HtIC) was the only pathotype which produced true conidia in submerged culture. HtIC began producing conidia after 3 days incubation reaching a peak ranging from 6.8 × 10⁵ to 9.7 × 10⁷ conidia/ml between 6 and 11 days. A concentration of 10 g/liter of corn steep liquor and 0.2% Tween 80 were essential for maximum conidiation. Submerged conidia had a smooth but somewhat rugose conidial walls, whereas aerially formed conidia were distinctly verrucose. Germination of submerged conidia ranged from 5.2 to 12.9% and were virulent causing 32.5% infection to adult citrus rust mites when sprayed on citrus foliage at 1.2 × 10⁹ conidia/ml.     

Blue Light and Abscisic Acid Independently Induce Heterophyllous Switch in Marsilea quadrifolia

In natural habitats Marsilea quadrifolia L. produces different types of leaves above and below the water level. In aseptic cultures growth conditions can be manipulated so that leaves of the submerged type are produced continuously. Under such conditions the application of either blue light or an optimal concentration of abscisic acid (ABA) induced the development of aerial-type leaves. When fluridone, an inhibitor of ABA biosynthesis, was added to the culture medium it did not prevent blue light induction of aerial leaf development. During blue light treatment the endogenous ABA level in M. quadrifolia leaves remained unchanged. However, after the plants were transferred to an enriched medium, the ABA level gradually increased, corresponding to a transition in development from the submerged type of leaves to aerial leaves. These results indicate that the blue light signal is not mediated by ABA. Therefore, in the regulation of heterophyllous determination, discrete pathways exist in response to environmental signals.     

Pyrene Metabolism in Crinipellis stipitaria: Identification of trans-4,5-Dihydro-4,5-Dihydroxypyrene and 1-Pyrenylsulfate in Strain JK364

The isolation and identification of two novel metabolites in the fungal metabolism of pyrene are described. The plant-inhabiting basidiomycete Crinipellis stipitaria JK364 metabolized pyrene, a polycyclic aromatic hydrocarbon containing four rings, when grown in submerged cultures in a medium containing malt extract, glucose, and yeast extract. In experiments with [¹⁴C] pyrene, after 7 days of incubation 40% of the labeled substrate was converted into organic solvent-extractable metabolites. Metabolites isolated from cultures grown with pyrene were identified as 1-pyrenylsulfate and trans-4,5-dihydro-4,5-dihydroxypyrene. 1-Hydroxypyrene, the precursor of 1-pyrenylsulfate, was also detected. 1-Pyrenylsulfate was isolated from mycelial extracts, whereas trans-4,5-dihydro-4,5-dihydroxypyrene was recovered from the culture filtrate. Identification of the compounds was based on their UV spectra, mass spectra, and nuclear magnetic resonance spectra. This is the first report on the detoxification of a polycyclic aromatic hydrocarbon by a plant-inhabiting basidiomycete. The occurrence of 1-pyrenylsulfate and trans-4,5-dihydro-4,5-dihydroxypyrene among fungal metabolites of pyrene is also new.