Modified Czapek Dox Research Articles

Tailored biolixiviation of spent LiCoO2 cathode batteries for sustainable metal recovery: Effects of consortia adaptation and particle size

With the rise of lithium-ion battery production, recycling valuable metals like Fe, Al, Cu, Ni, and Co is crucial. This study explores sustainable recovery from spent LiCoO2 cathode batteries via biolixiviation, focusing on microbial adaptation and particle size. Batteries were shredded into four size fractions, with mid-sized fractions (0.5–6 mm) showing the highest metal content and optimal microbial conditions. Microbial consortia were isolated using Modified Czapek-Dox (MCD) and M9 media. Metagenomic analysis revealed shifts in dominant bacteria, with Acidobacteria and Actinobacteria initially, and Chloroflexi, Firmicutes, and Proteobacteria later. Enzymatic profiling showed increased activity of key enzymes under metal-adapted conditions. Biolixiviation experiments revealed the highest metal recovery in mid-sized fractions at different conditions including the influence of microbial adaptation and the type of media used: Fe (47.2 %-53.0 %), Al (38.7 %-40.5 %), Cu (29.0 %-31.0 %), Ni (25.6 %-27.4 % in the largest fraction), and Co (40.1 %-41.0 %). Adapted consortia had higher recovery rates, offering a sustainable recycling route.

L-Asparaginase producing ability of Aspergillus species isolated from tapioca root soil and optimized ideal growth parameters for L-Asparaginase production

This research was designed to isolate the predominant L-asparaginase-producing fungus from rhizosphere soil of tapioca field and assess the suitable growth conditions required to produce maximum L-asparaginase activity. The Aspergillus tubingensis was identified as a predominant L-asparaginase producing fungal isolate from 15 isolates, and it was characterized by 18S rRNA sequencing. The L-asparaginase-producing activity was confirmed by pink color zone formation around the colonies in modified Czapek Dox agar plate supplemented with 1% L-Asparagine. The optimal growth conditions required for the L-asparaginase production by A. tubingensis were optimized as pH 6.0, temperature 30 °C, glucose as carbon source, 1.5% of L-Asparagine, ammonium sulphate as nitrogen source, rice husk as natural L-Asparagine enriched source, and 8 days of the incubation period. The L-Asparaginase activity from A. tubingensis was excellent under these optimal growth conditions. It significantly used rice husk as an alternative to synthetic L-Asparagine. As a result, this may be considered a sustainable method of converting organic waste into valuable raw material for microbial enzyme production.

Chitosan Modulates Volatile Organic Compound Emission from the Biocontrol Fungus Pochonia chlamydosporia.

Fungal volatile organic compounds (VOCs) are responsible for fungal odor and play a key role in biological processes and ecological interactions. VOCs represent a promising area of research to find natural metabolites for human exploitation. Pochonia chlamydosporia is a chitosan-resistant nematophagous fungus used in agriculture to control plant pathogens and widely studied in combination with chitosan. The effect of chitosan on the production of VOCs from P. chlamydosporia was analyzed using gas chromatography-mass spectrometry (GC-MS). Several growth stages in rice culture medium and different times of exposure to chitosan in modified Czapek-Dox broth cultures were analyzed. GC-MS analysis resulted in the tentative identification of 25 VOCs in the rice experiment and 19 VOCs in the Czapek-Dox broth cultures. The presence of chitosan in at least one of the experimental conditions resulted in the de novo production of 3-methylbutanoic acid and methyl 2,4-dimethylhexanoate, and oct-1-en-3-ol and tetradec-1-ene in the rice and Czapek-Dox experiments, respectively. Other VOCs changed their abundance because of the effect of chitosan and fungal age. Our findings suggest that chitosan can be used as a modulator of the production of VOCs in P. chlamydosporia and that there is also an effect of fungal age and exposure time.

Optimizing the culture conditions for L-Asparaginase production from endophytic fungus Curvularia sp. LCJ413 through conventional and statistical approach

L-Asparaginase (L-ASNase) is a crucial anti-tumour drug used to cure acute lymphocytic leukaemia. The current study aimed to enhance the production medium for the endophytic fungus Curvularia sp. LCJ413 that showed significant L-ASNase activity. L-ASNase production from Curvularia sp. LCJ413 was examined in six different media to select an appropriate liquid medium. Among the various media tested, Modified Czapek Dox broth (MCDB) exhibited the maximum L-ASNase activity (8.81 ± 0.52 U/mL). Physical (pH and temperature) and nutritional (carbon, nitrogen, inducer, and their concentrations) parameters were also optimized to boost L-ASNase production. Results of the study suggested a temperature of 28°C at pH 7 with 2 g/L maltose, 10 g/L L-Asparagine, and 25 g/L ammonium sulphate as the optimal carbon, inducer, and nitrogen source resulted in a high L-ASNase activity of 18.9 ± 0.40 U/mL. The statistical enhancement of L-ASNase by Response Surface Methodology (RSM) produced 20.11 U/mL of L-ASNase, which was 2.2 fold higher than the non-optimized medium. This is the first study on L-ASNase production from the endophytic Curvularia sp. LCJ413 isolated from Vitex negundo medicinal plant. Continuous fermentation with the medium composition provided in the study can produce L-ASNase on a large scale.

Cacti as low-cost substrates to produce L-asparaginase by endophytic fungi.

This study aimed to select endophytic fungi to produce L-asparaginase and partially optimising the production of the enzyme using cacti as substrate. Seventeen endophytes were assessed for intracellular enzymatic potential in modified Czapek Dox's medium using L-proline as an inducer. The best producer was evaluated for intracellular and extracellular enzymatic activity in modified Czapek Dox's medium using flours of Opuntia ficus-indica and Nopalea cochenillifera as substrate. The biomass and L-asparaginase production profile was analysed and the best conditions for enzyme production were verified using factorial design. Penicillium decaturense URM 7966, Diaporthe ueckerae URM 8321, and Colletotrichum annellatum URM 8538 produced 0.76 U g- 1, 0.87 U g- 1, and 0.74 U g- 1L-asparaginase, respectively. Diaporthe ueckerae URM 8321 produced only intracellular L-asparaginase, using flours of N. cochenillifera (0.72 U g- 1) and O. ficus-indica (0.90 U g- 1) and the last was selected for the next steps. The ideal time for biomass and L-asparaginase production was 120h. The best conditions for enzyme production (1.67 U g- 1) were initial pH 4.0, inoculum concentration 1% and cacti flour concentration 0.2%; where was observed an increase of 46.11% in compared to the initial production. Opuntia ficus-indica flour is indicated as an alternative low-cost substrate for the production of L-asparaginase by the endophytic fungus D. ueckerae URM 8321.

Statistical optimization of L-asparaginase production by Cladosporium tenuissimum

Background L-asparaginase produced by plant and bacteria can be used in the pharmaceutical and food industry. Unlike the bacterial counterparts, fungal L-asparaginase has more stability, more activity, and less adverse effects. Central composite design (CCD) was used to optimize temperature, pH, incubation time, and carbon-to-nitrogen ratio for L-asparaginase production by Cladosporium tenuissimum via submerged fermentation. CCD reduces the number of tests and time for optimization. Objective To optimize the culture conditions, such as temperature, pH, production time, and the ratio between concentration of carbon and nitrogen sources, for the production of L-asparaginase by isolated C. tenuissimum via submerged fermentation. Materials and methods Primarily, four significant parameters (temperature, pH, incubation period, and carbon-to-nitrogen ratio) were identified that affect the production process of L-asparaginase via submerged fermentation using the modified Czapek Dox medium. CCD was used to optimize the selected parameters concurrently, and their results were compared. Results and conclusions The highest L-asparaginase enzyme activity obtained was 2.6471 U/ml at 37°C, pH 6.2, incubation time 72 h, and 2 : 1 carbon-to-nitrogen ratio. The P value of interaction between every two factors was only significant for the interaction between temperature and incubation period (P

Production, optimization and partial purification of l-asparaginase from endophytic fungus Aspergillus sp., Isolated from Cassia fistula

Endophytes are relatively less studied microbes which in future may serve as potential sources of novel secondary metabolites for use in medical, agricultural and pharmaceutical industries. In present study six fungal endophytes were isolated from the leaves, stem and fruit of Cassia fistula and screened for asparaginase production on modified Czapek Dox agar supplemented with L-asparagine as a substrate and phenol red as indicator. L-asparaginase production by Aspergillus sp. using variable carbon and nitrogen sources, temperature and pH revealed maximum asparaginase production with xylose as carbon source and ammonium nitrate as nitrogen sources at pH 6.0 with incubation temperature of 30°C. The disc size optimization for enzyme revealed maximum activity with 10 mm size disc (0.22 IU mg−1 enzyme activity). The endophyte studied showed maximum activity under stationary condition (2.7 IU mg−1) than under shaking condition. Various purification steps included salt precipitation, dialysis, followed by separation on Sephadex G-100 gel filtration. The partially purified enzyme showed the activity of 4.9 IU mg−1. It may be concluded that the isolated Aspergillus sp. may serve as a vital source of L-asparaginase for future use.

Optimization of culture conditions and bench-scale production of anticancer enzyme L-asparaginase by submerged fermentation from Aspergillus terreus CCT 7693

L-Asparaginase amidohydrolase (EC 3.5.1.1) has received significant attention owing to its clinical use in acute lymphoblastic leukemia treatment and non-clinical applications in the food industry to reduce acrylamide (toxic compound) formation during the frying of starchy foods. In this study, a sequential optimization strategy was used to determine the best culture conditions for L-asparaginase production from filamentous fungus Aspergillus terreus CCT 7693 by submerged fermentation. The cultural conditions were studied using a 3-level, central composite design of response surface methodology, and biomass and enzyme production were optimized separately. The highest amount of biomass (22.0 g·L−1) was obtained with modified Czapek–Dox medium containing glucose (14 g·L−1), L-proline (10 g·L−1), and ammonium nitrate (2 g·L−1) fermented at 37.2 °C and pH 8.56; for maximum enzyme production (13.50 U·g−1), the best condition was modified Czapek–Dox medium containing glucose (2 g·L−1), L-proline (10 g·L−1), and inoculum concentration of 4.8 × 108 espore·mL−1 adjusted to pH 9.49 at 34.6 °C. The L-asparaginase production profile was studied in a 7 L bench-scale bioreactor and a final specific activity of 13.81 U·g−1 was achieved, which represents an increase of 200% in relation to the initial non-optimized conditions.

Bacillus subtilis UBTn7, a potential producer of L - Methioninase isolated from mangrove, Rhizophora mucronata

L-methioninase is an enzyme that degrades sulfur-containing amino acids to α-keto acids, ammonia, and thiols. L-methioninase could be found in plants, bacteria, and fungi. The aims of this study was to obtain L-methioninase-producing endophytic bacteria isolated from mangrove Rhizophora mucronata. The mangrove was collected from Jenu Beach, Tuban, East Java, Indonesia. The samples were roots, stems, and leaves of Rhizophora mucronata. Endophytic bacteria were pure isolated using LB agar medium. Each bacteria were screened its capability to produce L-methioninase using selective media namely modified Czapek Dox agar. The best producer of enzyme was further identified with morphological and biochemical analysis. The result showed that three bacteria produced L-methioninase. Based on the result of morphological and biochemical analysis, the best producer was Bacillus subtilis UBTn7.

Statistical optimization of L-asparaginase production by using Fusarium solani

Background and objective The enzyme l-asparaginase is important as a therapeutic agent in the treatment of acute lymphocytic leukemia. It has been observed that microorganisms such as yeast and filamentous fungi such as Aspergillus spp., Penicillium spp., and Fusarium spp. are commonly produced asparaginases with fewer adverse effects. Fusarium solani was selected to be the most potent microbial isolate for l-asparaginase production. The factors controlling l-asparaginase production, such as containing media, were implemented to increase the yield of l-asparaginase. Statistical experimental design such as the Plackett–Burman enables finding out the most effective factors that increase the yield of l-asparaginase. Materials and methods Aspergillus rubber, Aspergillus terreus, Epico niger, Penicillium cyclopium, and F. solani were chosen for screening the l-asparaginase activity using phenol red – a pH indicator. l-Asparagine and the modified Czapek Dox media were tested for the production of l-asparaginase from F. solani under different incubation times. To optimize l-asparaginase production by using F. solani, the combined effect of seven variables (l-asparagine, sucrose, NaNo3, K2HPO4, MgSO4, KCl, and time) were assessed using the central composite design. Results and conclusion All the four fungal strains screened for l-asparaginase activity showed positive results in the rapid plate assay method. The medium employed contained asparagine with phenol red, and after incubation, pink zones around the colonies were observed. F. solani was a potential source for a high yield of l-asparaginase enzyme and high substrate specificity. Modified Czapek medium II was the most suitable for l-asparaginase, showing an activity of 121 U/ml. The result on l-asparaginase activity according to the screening Plackett–Burman experiments gives a medium composed of the following (g/l): sucrose (1); l-asparagine (0.5); KH2PO4. (0.75), MgSO4 (0.7); H2O (0.72); KCl (0.72); (NH4)2SO4 (11.7) at an incubation time of 3 days. For further investigation, x1 K2HPO4, x2 sucrose, and x3 time were found to be the most significant variables affecting l-asparaginase activity. The second optimization step was to identify optimal values of the three factors that bring about maximum l-asparaginase activity, using the central composite designed experiment. The results showed that K2HPO4 (2.5 g/l), sucrose (4 g/l), and the time (8 days) were critical in the production of l-asparaginase.

Confined enzymatic activity of S. scitamineum and U. esculenta at the Smut Gall during Infection

We aimed to evaluate the enzymatic abilities of U. esculenta and S. scitamineum to degrade cellulose, pectin and lignin and explain the possible confinement of the smut - fungi in the smut-gall. S. scitamineum and U. esculenta were isolated from the smut gall of S. spontaneum and Z. latifolia plants, respectively. The teliospores were inoculated into a culture flask containing various concentrations (3%-10%) of pure cellulose powder to check the degradation ability of the fungi. The fungi were sub-cultured onto modified Czapek Dox Agar (CDA) supplemented with 0.3% carboxy methyl cellulose (CMC) followed by Gram’s Iodine test to determine the relative cellulolytic activity index (RCAI). Extracellular proteins were harvested and profiled by SDS-PAGE and subsequently zymography was carried out with regular 0.1% substrate infiltration to determine cellulolytic, pectinolytic and laccase activities. The fungi were capable of producing cellulase and pectinase but lacked the laccase enzyme required to degrade the lignin, perhaps as an essential evolutionary requirement for the completion of their life cycle in the host. Importantly, the lack of laccase activity by U. esculenta and S. scitamineum could partially explain why the fungal colonization remains localized in the topmost part of S. spontaneum and Z. latifolia.

Isolation and screening of L-asparaginase free of glutaminase and urease from fungal sp.

l-Asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukaemia (ALL), a malignant disorder in children. l-Asparaginase helps in removing acrylamide found in fried and baked foods that is carcinogenic in nature. l-Asparaginase is present in plants, animals and microbes. Various microorganisms such as bacteria, yeast and fungi are generally used for the production of l-asparaginase as it is difficult to obtain the same from plants and animals. l-Asparaginase from bacteria causes anaphylaxis and other abnormal sensitive reactions due to low specificity to asparagine. Toxicity and repression caused by bacterial l-asparaginase shifted focus to eukaryotic microorganisms such as fungi to improve the efficacy of l-asparaginase. Clinically available l-asparaginase has glutaminase and urease that may lead to side effects during treatment of ALL. Current work tested 45 fungal strains isolated from soil and agricultural residues. Isolated fungi were tested using conventional plate assay method with two indicator dyes, phenol red and bromothymol blue (BTB), and results were compared. l-Asparaginase activity was measured by cultivating in modified Czapek–Dox medium. Four strains have shown positive result for l-asparaginase production with no urease or glutaminase activity, among these C7 has high enzyme index of 1.57 and l-asparaginase activity of 33.59 U/mL. l-Asparaginase production by C7 was higher with glucose as carbon source and asparagine as nitrogen source. This is the first report focussing on fungi that can synthesize l-asparaginase of the desired specificity. Since the clinical toxicity of l-asparaginase is attributed to glutaminase and urease activity, available evidence indicates variants negative for glutaminase and urease would provide higher therapeutic index than variants positive for glutaminase and urease.

Purification, Kinetic Properties and Antitumor Activity of L-Glutaminase from Penicillium brevicompactum NRC 829

Aim: The aims of the present study were to purify and characterize L-glutaminase from Penicillium brevicompactum NRC 829; and to evaluate the antitumor activity of the purified enzyme against different tumor human cell lines. Study Design: Testing of antitumor activity of L-glutaminase, purified from a filamentous fungal strain, against four different tumor human cell lines. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2011 and February 2012. Methodology: P. brevicompactum NRC 829 was grown and maintained on modified Czapek Dox agar (MCD) medium. Cell-free extract was directly used as the source of crude enzyme. L-glutaminase was purified by heat treatment for 20 min at 50oC, followed by gel filtration on Sephadex G-100 and G-200 columns. Results: An intracellular L-glutaminase from Penicillium brevicompactum NRC 829 was purified to homogeneity (162.75 fold) with an apparent molecular mass (Mr) of 71 kDa. The purified enzyme showed its maximal activity against L-glutamine when incubated at pH 8.5 Research Article British Microbiology Research Journal, 4(1): 97-115, 2014 98 at 50oC for 30 min. The purified enzyme retained about 92 % of its initial activity after incubation at 70oC for 30 min indicating the thermo-stability nature of this enzyme. The highest activity was reported towards its natural substrate, L-glutamine, with an apparent Km value of 1.66 mM. The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 63.3μg/ml. Conclusion: L-glutaminase purified from Penicillium brevicompactum NRC 829 is a potential candidate in food and pharmaceutical industries.

A New Native Source of Tannase Producer, Penicillium sp. EZ-ZH190: Characterization of the Enzyme

Background: Tannase can be obtained from the various sources for example tannin rich plants; however microbial sources are preferred for industrial production. In microbial sources, the Aspergillus and Penicillium genus and lactic acid bacteria mostly produce tannase. However, it has been identified that this enzyme is produced by many fungi and bacteria, but researches are continuing to find new species. Objectives: The aim of this study was to isolate a tannase-producing fungi from moldy tea leaves and to study some properties of its enzyme. Materials and Methods: The present study was done via two steps. At first, industrially important tannaseproducing fungi were isolated from moldy tea leaves using the simple agar plate method followed by the screening of organisms capable of producing tannase using the enrichment culture technique in modified Czapek Dox’s agar. Finally, tannase obtained from the best isolate was partially purified and characterized. Results: Tannase produced by Penicillium sp. EZ-ZH190 isolated from moldy tea leaves was partially purified and characterized. Maximum enzyme production (4.33 U.mL-1) was recorded after 96 hours of incubation at 30 °C in submerged culture (100 rpm) utilizing 1% (w/v) tannic acid as a sole carbon source. This tannase exhibited optimum activity at 35 °C and at pH of 5.5, and showed nearly 50% of its maximal activity at 50 °C. In the present study, tannase from Penicillium sp. EZ-ZH190 had KM and Vmax values of 1.24 mM and 17.09 U.mL-1, respectively, and showed more than 50% stability at salt (NaCl) concentration of 1 M for 24 hours. Conclusions: Tannase productivity of Penicillium sp. EZ-ZH190 (0.045 U.mL-1.h-1) is comparable with the maximum tannase productivity in the reported literatures, and the biochemical characteristics showed by Penicillium sp. EZ-ZH190 tannase are considered favorable for tannin biodegradation in the industry. So, we concluded that Penicillium sp. EZ-ZH190 is a good strain for use in the efficient production of tannase.

KINETIC STUDIES OF L-ASPARAGINASE FROM Penicillium digitatum

L-Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and other related malignancies. Its further use includes reduction of asparagine concentration in food products, which may lead to formation of acrylamide. Currently bacterial asparaginase is produced at industrial scale, but the enzyme isolated from bacterial origin is often associated with adverse reactions. These side effects require development of asparaginase from alternative sources. In the present study, Penicillium digitatum was explored for the production of extracellular L-asparaginase using modified Czapek–Dox media. The enzyme was purified about 60.95-fold and then kinetic study showed that the Km value of the enzyme was 1 × 10−5 M. The optimum pH and temperature for the enzyme were 7.0 and 30°C, respectively. The optimum incubation period for L-asparaginase was 15 min. This work concludes that this enzyme can be a suitable candidate due to its strong kinetic properties, and further research can usher into development of asparaginase formulation from fungal origin with less adverse effects.

Production of tannase through solid state fermentation using Indian Rosewood (Dalbergia Sissoo)sawdust—a timber industry waste

The tannase producing strain Aspergillus heteromorphus MTCC 8818 was used in the present study for the production of tannase under solid state fermentation using Rosewood (Dalbergia sissoo) sawdust—a timber industry waste—as substrate. Various physico-chemical parameters were optimized for extracellular yield of tannase. Maximum tannase (1.84 U/g dry substrate) and gallic acid (5.4 mg/g ds) was observed at 30 °C after 96 h of incubation. Czapek dox medium was found to be the best moistening agent, with pH and relative humidity of 5.5 and 70 %, respectively. The constituents of Czapek dox medium were varied to enhance enzyme production. The optimum concentration of modified Czapek dox constituents contained 0.2 % NaNO3, 0.05 % K2HPO4 and MgSO4, 0.15 % KCl. Among the additional salts supplemented to Czapek dox medium, ZnSO4 and CuSO4 were found to have a stimulating effect, with a relative tannase activity of 116 and 111 %, respectively. Glucose as an external carbon source was found to be a repressor of enzyme production.

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.

Effects of ambient pH and nutritional factors on antifungal activity of the mycoparasite Coniothyrium minitans

Antifungal activity (AA) of the mycoparasite Coniothyrium minitans (CM) against Sclerotinia sclerotiorum (SS), designated as AA CM–SS, was detected by measuring the effect of cell-free cultural filtrates of CM on inhibition of growth (IG) of SS on potato dextrose agar. Using this bioassay method, effects of ambient pH and nutritional factors on AA CM–SS in CM cultures grown in various liquid media were investigated. Results showed that production of AA CM–SS by CM was closely related to ambient pH. After incubation for 9–15 days, AA CM–SS was high in modified Czapek–Dox broth (MCD) with IG values being 78–90%, but low in potato dextrose broth (PDB) with IG values being 9–18%. In contrast, pH value was as low as 3.3–3.6 in the MCD cultures, whereas as high as 6.0–7.2 in the PDB cultures. In the pH-buffered experiment, AA CM–SS was detected in MCD cultures of CM buffered at pH 2–8 with the optimum pH being 3–5, under which AA CM–SS in PDB cultures of CM was greatly enhanced with the IG values reaching up to 46–67%. Results also indicated that AA CM–SS in CM cultures can be affected by nutritional factors and their effects were also related to ambient pH. In the basal medium Czapek mineral salts, most carbon and nitrogen sources investigated were favourable to CM to produce AA CM–SS with the IG values being 73–91% and the final pH values being 2.7–4.1. In the other basal medium potato extracts, a few carbon and nitrogen sources were favourable for CM to produce AA CM–SS with the IG values being 40–60% and the final pH values being 2.7–5.5. In the pH-buffered experiments with selected carbon and nitrogen sources, or with mycelial or sclerotial extracts of SS, AA CM–SS was much higher in CM cultures buffered at pH 3 than that buffered at pH 6. Additional experiments showed that AA CM–SS could be induced by the phytotoxin oxalic acid (OA), but not by sodium oxalate, further suggesting the importance of acidic pH in production of AA CM–SS by CM. Meanwhile, leaf blight incidence on Chinese cabbage caused by SS was greatly reduced in the treatment of CM conidia pre-germinated in PDB plus OA (16 mM) than that in the treatment of CM conidia pre-germinated in PDB alone. This study suggests that ambient pH plays an important role in regulation of the antifungal activity of CM against SS.

Suppression of Sclerotinia sclerotiorum by antifungal substances produced by the mycoparasite Coniothyrium minitans

A study was conducted to investigate production of antifungal substances (AFS) by Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in modified Czapek-Dox (MCD) broth and potato dextrose broth (PDB), and effects of AFS of Cm on mycelial growth and germination of sclerotia and ascospores of Ss and incidence of leaf blight of oilseed rape caused by Ss. Results showed that mycelial growth of Ss was reduced by 41.6 and 84.5% on 3 day-old cultures grown on potato dextrose agar (PDA) amended with 10% (v v−1) of cultural filtrates of Cm grown in MCD (MCDcm) after incubation for 6 and 15 days, respectively, and by 2.7 and 15.7% on PDA amended with 10% (v v−1) of cultural filtrates of Cm grown in PDB for 6 and 15 days, respectively. In addition to retardation of mycelial growth, morphological abnormality of Ss such as hyphal swellings and cytoplasm granulation were also observed in colonies grown on PDA amended with cultural filtrates of MCDcm. Sclerotia of Ss soaked in the filtrates of MCDcm for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA was delayed, compared to sclerotia treated with MCD. Germination of ascospores of Ss was unaffected on PDA amended with 10% of the filtrates of MCDcm. However, germ tubes of Ss were shortened and deformed by the formation of hyphal swellings in the treatment of MCDcm. Treatment of leaves of oilseed rape with cultural filtrates of MCDcm reduced incidence of leaf blight caused by Ss, compared to the controls (water or MCD). This study suggests that AFS produced by Cm plays an important role in the suppression of mycelial growth and germ-tube development of ascospores of Ss and that there is potential for using AFS of Cm to control leaf blight of oilseed rape caused by ascospores of Ss.

Optimization of tannase biosynthesis by a newly isolated Rhizopus oryzae

A strain isolated locally and identified as Rhizopus oryzae (RO, IIT KGP) was found to synthesise an extracellular enzyme, tanin acyl hydrolase, showing its degradability of tannic acid to gallic acid. For maximizing the enzyme secretion in the fermented broth, the influencing parameters were optimized in shake flask culture. Experiments showed that modified Czapek dox medium with 2% tannic acid, 1% glucose, 0.05% sodium nitrate incubated for 4 days with 2 days old inoculum was the optimum for the synthesis of tannase by Rhizopus oryzae (RO, IIT KGP). Maximum enzyme activity was found to be 6.12 U/ml.