Formation and Regeneration of Protoplasts of the Actinorhizal Nitrogen-Fixing Actinomycete Frankia

Procedures for forming and regenerating protoplasts of four Frankia strains are described. Cells obtained from growth medium containing 0.1% glycine were digested with lysozyme (250 μg/ml) in a medium containing 0.5 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂. Protoplasts were formed during 15 to 120 min of digestion at 25°C. Optimum conditions for protoplast regeneration involved placing protoplasts on a layer of complex growth medium containing 0.3 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂ which was overlaid with a layer of 0.8% low-melting-point agarose containing 0.5 M sucrose, 5.0 mM MgCl₂, and 5.0 mM CaCl₂. The maximum regeneration efficiency was 36.9% for strain CpI1, 1.3% for strain ACN1^AG, 27% for strain EAN1pec, and 20% for strain EuI1c.     

Preliminary determination of mycotoxin binding to soil when leaching through soil with water

Mycotoxin-contaminated crops that are left in the field are potential contaminants of groundwater. Aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) distribution in soil-water systems and the comparative response of aflatoxin-contaminated corn and pure aflatoxin when leached through soil were investigated using columns. Each experiment was repeated once. Eluates and soil extracts were analyzed for AFB₁ and its metabolites and FB₁ along with different amounts of pure FB₁ and water mixtures. AFB₁ was detected in water samples from columns containing 10% and 20% silty clay loam soil and aflatoxin-contaminated corn mixtures and in the upper (top) 2.5 cm of soil from the 10 cm soil column. Aflatoxin B₂ (AFB₂) was detected in eluates from the column containing 10% soil and aflatoxin-contaminated corn mixture and from the column containing aflatoxin-contaminated corn alone. No AFB₂ was detected in eluates from the column containing 20% soil and aflatoxin-contaminated corn mixture. No detectable amount of aflatoxin was observed in eluates from the containing 50% silty clay loam soil and aflatoxin contaminated corn. No detectable amount of FB₁ was observed in eluates or soil extracts, but FB₁ was detected in the mixtures of pure FB₁ and water.     

Bacterial phylogenetic diversity in a Spartina marsh in China

The bacterial phylogenetic diversities of the bacterial communities in the salt marsh colonized by Spartina alterniflora (SA) and uncultured marsh (UM) along the Yellow Sea of China were analyzed based on the 16S rDNA PCR techniques. Two libraries containing 251 clones of 16S rRNA genes from the Spartina colonized marsh and 283 from UM were constructed by PCR using a bacteria-specific primer 8f and the common primer 1542r. Forty-seven clones from SA and 55 from UM were selected for partial sequencing. A phylogenetic tree was constructed by the alignment analysis of the total environmental DNA sequences. Clones were clustered into 8 divisions of bacteria: (1) Proteobacteria containing 12 genera in beta, gamma, delta and epsilon subdivision; (2) Bacteroidetes containing 7 genera; (3) Planctomycetes containing 2 genera; (4) Firmicutes containing 1 genus; (5) Spirochaetes containing 1 genus; (6) Acidobacteria containing 1 genus; (7) Actinobacteria containing 1 genus; and (8) BRC1 containing 1 genus, as well as 3 groups of bacteria that could not be clustered into any recognized bacterial divisions or candidate divisions. BLAST searches of the GenBank database confirmed that 79.7% of the clones in the two libraries were closely related to the 16S rDNA sequences of the uncultured bacteria with the similarity ≥90%. Among them, Proteobacteria (36.7% and 54.4% of the total clones from the SA and UM libraries, respectively) and Bacteroidetes (30.0% and 18.3%, respectively) are two dominant groups in both the sites. However, some phyla, such as Firmicutes and Actinobacteria, were found at SA site but not at UM site, and vice versa, some phyla, such as Spirochaetes and BRC1, were found at UM, but not at SA. Moreover, the dominant species in each phylum were varied, dependent on whether or not the marsh was colonized by Spartina. For instance, in Bacteroidete, 44.4% clones at SA library were affiliated to Gelidibacter, but 32.7% clones were affiliated to Flexibacter at UM library. In Proteobacteria, the clones at UM library affiliated to δ, γ, ? and β-subdivisions were 38.8%, 34.7%, 23.5% and 3.1%, respectively, but at SA library were 30.3%, 47.0%, 13.6% and 9.1%, correspondingly. These results indicated that the bacterial diversity in the salt marsh along the Yellow Sea of China was greatly changed after Spartina colonization.     

Laboratory and field evaluation of commercial repellent formulations against mosquitoes (Diptera: Culicidae) in Queensland, Australia

Abstract  Laboratory tests of commercial repellent formulations were conducted against Anopheles farauti Laveran, Culex annulirostris Skuse, Ochlerotatus vigilax (Skuse) and Stegomyia aegypti (L.). The majority of repellent formulations tested contain N,N ,-diethyl- 3 -methylbenzamide (also known as diethyl- m -toluamide, commonly called deet). Two formulations containing picaridin (1-piperidinecarboxylate acid, 2-(2-hydroxyethyl)-1-methylpropylester, also known as KBR 3023), one containing ethyl butylacetylaminopropionate (EBAP) and two formulations containing essential oils (e.g. Citronella oil) were also tested. In the laboratory tests, repellent formulations containing deet provided the best protection, and picaridin and EBAP also provided good protection. Citronella oil provided only limited protection. Two field trials to compare commercially available repellent formulations containing picaridin and deet against mosquitoes at Redcliffe, Queensland, were conducted. In the first, Autan Repel, containing 9.3% picaridin, RID, containing 10% deet, and Bushman Ultra, containing 80% deet in a gel, were compared. In the second, Autan Repel Army 20, containing 19.2% picaridin, OFF! Skintastic, containing 7% deet, and Aerogard, containing 12% deet, were compared. The predominant mosquito in both tests was Cx. annulirostris . Bushman provided >95% protection against all mosquitoes for at least 8 h when tests ceased. The other deet repellents also provided good protection against mosquitoes, with RID providing 5 h, Skintastic 4 h and Aerogard 2 h protection. The Autan repel (9.3% picaridin) provided >95% protection for 3 h, and Autan Repel Army (19.2% picaridin) provided 4 h protection. These studies have shown that commercial formulations of both deet and picaridin provide good protection against Cx. annulirostris , an important vector of arboviruses in Australia.     

Escape of Steinernema feltiae from Alginate Capsules Containing Tomato Seeds

The entomogenous nematode Steinernema feltiae was encapsulated in an alginate matrix containing a tomato seed. When these capsules were placed on 0.8% agar for 7 days, the seed germinated and ca. 20% of the nematodes escaped from the capsules, whereas only 0.1% escaped from capsules without seeds. When capsules containing nematodes and a seed were planted into sterilized or nonsterilized soil, nematodes escaped to infect Galleria mellonella larvae. When seed in capsules containing ca. 274 nematodes per capsule were planted in nonsterilized soil, Galleria mortality was 90% 1 week later. Galleria mortality declined to 27%, 23%, and 0% in weeks 2, 4, and 8 postplant, respectively. In sterilized soil, Galleria mortality was 96% and did not differ significantly from the nonsterilized soil in week 1, but was significantly higher in sterilized soil over nonsterilized soil for week 2 (81%) and week 4 (51%). When capsules containing nematodes only were used, Galleria mortality was 71% in sterilized soil 1 week after planting and 58%, 33%, and 12% in weeks 2, 4, and 8 postplant, respectively. In nonsterilized soil, Galleria mortality was 8%, 30%, 21%, and 28% after 1, 2, 4, and 8 weeks, respectively, using only encapsulated nematodes. When the number of nematodes per capsule was increased to ca. 817, Galleria mortality was 92 % or higher in sterilized soil from week 1 to week 4.     

Bacterial degradation of vinyl chloride

Summary Mycobacterium L1 can grow on vinyl chloride as sole carbon and energy source. Application of this bacterium to remove vinyl chloride from waste gases is proposed. From air containing 1% vinyl chloride 93% of the vinyl chloride was removed by passing the air through a fermentor containing a growing population ofMycobacterium L1.     

Disinfection of the Skin: An Assessment of Some New Preparations

Repeated hand washing with a detergent solution containing 0·75% chlorhexidine digluconate was found to cause a large reduction in the resident skin flora which was slightly though significantly smaller than that caused by the use of 3% hexachlorophane liquid soap containing a phenolic preservative, chlorocresol 0·3%. Both agents caused a greater immediate reduction of bacteria after a single hand washing than the hexachlorophane liquid soap without a phenolic additive had shown in earlier experiments; the soap base containing chlorocresol 0·3% but no hexachlorophane was also found to cause a large reduction in skin flora. The chlorhexidine detergent solution had no residual disinfectant action on the skin after rinsing and drying the hands.Disinfection of an operation site for two minutes with povidone-iodine containing 1% available iodine in 70% ethyl alcohol caused about as great a reduction in resident flora as a similar treatment with alcoholic 0·5% chlorhexidine. Both treatments were more effective than disinfection with aqueous 1% cetrimide or 0·1% benzalkonium chloride solutions.     

Mannosylglycerate is essential for osmotic adjustment in Thermus thermophilus strains HB27 and RQ-1

We disrupted the mpgS encoding mannosyl-3-phosphoglycerate synthase (MpgS) of Thermus thermophilus strains HB27 and RQ-1, by homologous recombination, to assess the role of the compatible solute mannosylglycerate (MG) in osmoadaptation of the mutants, to examine their ability to grow in NaCl-containing medium and to identify the intracellular organic solutes. Strain HB27 accumulated only MG when grown in defined medium containing 2% NaCl; mutant HB27M9 did not grow in the same medium containing more than 1% NaCl. When trehalose or MG was added, the mutant was able to grow up to 2% of NaCl and accumulated trehalose or MG, respectively, plus amino acids. T. thermophilus RQ-1 grew in medium containing up to 5% NaCl, accumulated trehalose and lower amounts of MG. Mutant RQ-1M1 lost the ability to grow in medium containing more than 3% NaCl and accumulated trehalose and moderate levels of amino acids. Exogenous MG did not improve the ability of the organism to grow above 3% NaCl, but caused a decrease in the levels of amino acids. Our results show that MG serves as a compatible solute primarily during osmoadaptation at low levels of NaCl while trehalose is primarily involved in osmoadaptation during growth at higher NaCl levels.     

The relationship between ACE inhibitory activity and degradations of sulfur containing materials in Dolsan leaf mustard juice

This study was carried out to investigate the relationship ACE inhibitory activity and degradations of sulfur containing materials in Dolsan leaf mustard juice (DLMJ). The changes of sulfur containing materials which were treated with autolysis, myrosinase, ascorbate and papain were studied, as well as the changes of ACE inhibitory activity in DLMJ. At 37°C, sulfur containing materials by autolysis decreased most rapidly from 0.43% to 0.13% in the second day. Conversely, ACE inhibitory activity increased most from 66% to 87%, in the second day at 37°C. As myrosinase concentrations increased more, sulfur containing materials in DLMJ decreased more. The ACE inhibitory activities at 0, 0.5, 1, 2, and 4 Units of myrosinase for 240 min later were 70, 74, 75, 82, and 85%, respectively. At 1 mM ascorbate, concentrations of sulfur containing materials in DLMJ decreased more significantly on the second day than on the other days. At 1 mM ascorbate for 6 days, ACE inhibitory activity reached a maximum of about 92%. And, an increase of papain concentration was noted in accordance with a decreased sulfur containing materials. The maximum rate of ACE inhibitory activity at control, 3, 6, and 12 Units of papains treatments was shown as 70, 70, 75, and 78% at 60 min, respectively. These results suggested that the degradation of sulfur containing materials led to the increase of ACE inhibitory activity. Consequently, it was suggested that ACE inhibiting was significantly related to the degradatives of sulfur containing materials.     

小果卫矛愈伤组织诱导及植株再生体系的建立

以小果卫矛嫩茎为外植体，采用L₉（3⁴）正交设计法，研究了不同灭菌组合、不同基本培养基、不同浓度6-BA、2，4-D、NAA配比对小果卫矛愈伤组织诱导、再分化及生根的影响。结果表明：小果卫矛嫩茎最适的灭菌组合为75%酒精消毒30 s+0.1%升汞消毒15 min，愈伤组织诱导的最佳培养基为MS+3.0 mg·L^-1 6-BA+1.0 mg·L^-1 2，4-D，诱导率为79%；再分化的最佳培养基为MS+6-BA 2.0 mg·L^-1+NAA 0.2 mg·L^-1，再分化率为78.83%，1/2 MS+NAA 1.2 mg·L^-1适用于生根培养，生根率达到83.23%。     

First isolation and structural determination of cyclic beta-(1-->2)-glucans from an alga, Chlorella pyrenoidosa

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.     

Effects of Fusarium moniliforme culture extracts and fumonisin B1 on DNA,RNA and protein synthesis by baby hamster kidney cells

Baby hamster kidney cells (BHK-21) were exposed to culture filtrates of 4 Fusarium moniliforme isolates containing varying levels of fumonisin B1 (FMB1) and the effects upon RNA, DNA and protein synthesis were monitored. Cells were also grown on medium amended with FMB1 only for comparison. After 24 h incubation FMB1 (100 μg/100 ml medium) reduced protein synthesis by 4% and by 18% after 48 h. Culture filtrates containing the highest levels of FMB1 also caused the greatest inhibition in protein synthesis after 24 h but after 48 h protein synthesis levels were the same as controls even though the FMB1 level was 360 μg/100 ml. Only FMB1 reduced DNA synthesis, by 8% after 24 h but after 48 h DNA levels had increased by 40 % over controls. The culture filtrates containing the highest levels of FMB1 (360 μg/100 ml) reduced DNA synthesis more than 50% after 24 h and 48 h. Culture filtrates containing lesser amounts of FMB1 in some instances stimulated DNA synthesis and inhibited it in others. There was also no correlation in the level of FMB1 with the inhibition of RNA synthesis by BHK cells. It appears that metabolites other than fumonisin produced by F. moniliforme in culture can affect and both stimulate and inhibit RNA, DNA and protein synthesis by BHK cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation ofBoswellia serrata Roxb

Anin vitro procedure for large scale multiplication ofBoswellia serrata Roxb. has been developed using cotyledonary node segments. In average 4 shoots per node were obtained on Murashige and Skoog’s (MS) medium containing 0.5 mg dm^?3 6-benzylaminopurine (BAP) and 0.05 mg dm^?3 napthaleneacetic acid (NAA) within 22 d. By repeated subculture technique 90–100 shoots per node could be obtained after 88 d of initial culture. Shoots could be rooted on MS medium containing 1/4 salts, 1% saccharose, and a combination of 0.5 mg dm^?3 indole-3-butyric acid (IBA) and 0.25 mg dm^?3 indole-3-acetic acid (IAA). Addition of antioxidants like polyvinylpyrrolidone (PVP-50 mg dm^?3) and ascorbic acid (100 mg dm^?3) in both multiplication and rooting media prevented browning of cultures. Approximately 80% of shoots rooted within 8–10 d. Rooted plantlets were kept for 15 d in culture bottles containing Soilrite^TM irrigated with a nutrient solution containing 1/4 MS salts and finally transferred to pots containing soil: Soilrite^TM (1∶1), mixture with 70% transplantation success.     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-d (1 mg 1^-1), NAA (0.1 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1^-1) and BAP (1 mg 1^-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1^-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1^-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin     

Effects of temperature and dietary sucrose concentration on respiration in the silverleaf whitefly, Bemisia argentifolii

A system consisting of a flow-through chamber connected to a commercial infrared gas analysis system was developed to measure homopteran respiration during feeding. Using this system, respiration rates of 202 and 206 μmol CO(2) h(-1) g(-1) (4.96 and 5.04 ml CO(2) h(-1) g(-1)) were determined for whiteflies and cotton aphids, respectively, at 25 degrees C on diets containing 15% sucrose. These rates were considerably higher than those of other stationary insects, indicating that whiteflies and aphids maintain a relatively high metabolic rate when feeding. Whitefly respiration increased with temperature from 25 to 46 degrees C with a Q(10) of about 2 on diets containing 10, 15 and 20% sucrose, but less than 2 on diets containing 2.5 and 5% sucrose. Respiration rates were similar on the diets containing >10% sucrose, but were generally lower on the diets containing <10% sucrose. Respiration rates decreased upon extended exposure to 47 degrees C; the rate of decrease was inversely related to the dietary sucrose concentration up to 15%. The results indicate that whiteflies require a sucrose concentration of between 5 and 10% (i.e. 0.15 and 0.3 M) for maximum rates of metabolism while feeding. Higher concentrations of sucrose in the diet delayed high-temperature mortality, possibly a reflection of the high sucrose requirement for sorbitol synthesis in whiteflies.     

Protoplast isolation,culture and regeneration from Egyptian cultures of Pea and Beans

Abstract

A protocol of protoplast isolation from Egyptian varieties of pea and bean is reported. Protoplast cultures were established from apical shoots of pea (Pisum sativum) and suspension cultures of bean (Phaseolus vulgaris). To isolate protoplasts of pea, apical shoot tissues were digested for 10 h using enzyme solution containing 1% pectinase, 0.5% cellulase, 0.5% hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. For protoplast isolation from suspension culture of bean, collected cells were incubated for 6 h in digestion solution containing 0.5% pectinase, 0.25% of each of cellulase and hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. Purified protoplasts were cultured in liquid culture medium. Microcalli were obtained after 30 days of culture. Calli colonies with a diameter of about 5 mm were developed after one month of culturing on solid B5 medium containing 2% sucrose, 2 g/l casein hydrolysate, 0.7% agar and supplemented with either 1 mg/l of each 2,4-D and kin in case of pea or 2 mg/l 2,4-D+0.5 mg/l kin in case of bean. Protoplast derived callus of pea was successfully differentiated into shoot and root, and highest frequency of shoot organogenesis was recorded on medium containing 0.5 mg/l NAA+2 mg/l BA. Protoplast derived callus of bean, on the other hand, gave rise to a high frequency of root formation when cultured on medium containing 1 mg/l NAA, but attempts to regenerate shoots from this callus was unsuccessfull.     

A polyhydroxyalkanoates bioprocess improvement case study based on four fed-batch feeding strategies

The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl-PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l⁻¹ in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l⁻¹ in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l⁻¹ containing 65% PHA at the end of a 25-h incubation.     

Cuticle-Degrading Proteases Produced by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Their Induction in Different Media

Protease production by fourteen M. anisopliae isolates differing in geographical origin and host insect were investigated. Highest protease activity was observed during 4–8 days of culture incubation. Pr1 and Pr2 activity was investigated in various media containing different carbon and nitrogen source to evaluate the induction mechanism of these enzymes. Basal levels of Pr1 and Pr2 activity were observed in minimal medium suggesting constitutive production. Casein (1%) as an exogenous protein supplement was not able to induce significant release of Pr1 and Pr2 enzymes, whereas high levels of activity were observed in the medium containing colloidal chitin (2%) as sole carbon and nitrogen source. The pH, ammonia and oxalic acid production in in vitro conditions was also investigated and the alteration in pH for protease production was not significant in the different media used except for the medium containing casein (1%) as a supplement.     

Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III

By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities

Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies showing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml⁻¹, measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of β-glucosidase per gram of wheat bran.