Czapek Dox Broth Research Articles

Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp. isolated from Ipomoea carnea

An endophytic fungus displaying considerable antimicrobial activity was isolated from stem tissue of an invasive plant species, Ipomoea carnea. The fungus was identified as Quambalaria sp. and confirmed by ITS rDNA sequence analysis. A BLAST search result of the sequence indicated 97 % homology with Quambalaria cyanescens. Crude metabolites of the fungus showed considerable antimicrobial activity against a panel of clinically significant microorganisms. The metabolites showed highest in vitro activity against Shigella dysenteriae followed by Escherichia coli and Candida albicans. Optimum metabolites production required neutral pH and a 15-day incubation period. Bark extracts amended with fungal media demonstrated higher antimicrobial activity. Optimum metabolites activity was recorded in Czapek Dox broth amended with leaf extracts (CDB + LE) of the host plant. The metabolites showed UV λ-max in ethyl acetate at 284.6 nm with an absorbance value of 1.093. Phylogenetic tree generated by the Maximum Parsimony method showed clustering of our isolate with Q. cyanescens with supported bootstrap of 65 %. Species of Quambalaria are pathogens to Eucalyptus and occurrence of this fungus as endophytes support it to be a latent pathogen. Sequence base analysis and RNA secondary structure study also confirmed such a relationship. Secondary structural features like two hinges and a 5’ dangling end were found to be unique to our isolate. These structural features can also be used as potential barcodes for this fungus. The findings indicate that invasive plant species can be a reliable source of novel endophytes with rich antimicrobial metabolites. The study also validates the assumption that endophytes can become parasites and share a close affinity.

Lethal Effects ofAspergillus nigeragainst Mosquitoes Vector of Filaria, Malaria, and Dengue: A Liquid Mycoadulticide

Aspergillus niger is a fungus of the genus Aspergillus. It has caused a disease called black mold on certain fruits and vegetables. The culture filtrates released from the A. niger ATCC 66566 were grown in Czapek dox broth (CDB) then filtered with flash chromatograph and were used for the bioassay after a growth of thirty days. The result demonstrated these mortalities with LC50, LC90, and LC99 values of Culex quinquefasciatus 0.76, 3.06, and 4.75, Anopheles stephensi 1.43, 3.2, and 3.86, and Aedes aegypti 1.43, 2.2, and 4.1 μl/cm2, after exposure of seven hours. We have calculated significant LT90 values of Cx. quinquefasciatus 4.5, An. stephensi 3.54, and Ae. aegypti 6.0 hrs, respectively. This liquid spray of fungal culture isolate of A. niger can reduce malaria, dengue, and filarial transmission. These results significantly support broadening the current vector control paradigm beyond chemical adulticides.

<i>Aspergillus Niger</i> Metabolites Efficacies Against the Mosquito Larval (<i>Culex Quinquefasciatus</i>, <i>Anopheles Stephensi</i> and <i>Aedes Aegypti</i>) Population after Column Chromatography

Problem statement: Mosquito is a very common vector of several tropical diseases such as malaria, filariasis, dengue and chikungunya. Till todate, many xenobiotics have been used to control mosquito population and protect people from mosquito born diseases but increasing rate of resistance against these xenobiotics is a major concern in the disease control management. In contrast, extensive use of these xenobiotic may lead to cause opurtunistic disease like cancer. Therefore, biological control can thus be an effective and environmental friendly approach, which can be used as an alternative to minimize the mosquito population. Fungi and fungus-derived products are highly toxic to mosquitoes, yet have low toxicity to nontarget organisms. Aspergillus niger is a cosmopolitan fungus which is primarily isolated from soil and decomposing plant materials. Approach: In the present investigation, this fungus was cultured on Czapek Dox Broth (CDB) for extracellular metabolites production. The A. niger was maintained at 25°C under 75 ± 5% humidity for 15 days. The extracellular metabolites were filtered through Whatman no.-1 filter papers. The purification of crude metabolites was done with different ratio of ethanol and metabolites (ethanol: metabolites = 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9, respectively) using column chromatography. All fractions were applied for larvicidal efficacy against the larvae of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. The larvicidal efficacy was performed at six-log concentrations for 24, 48 and 72h. Results: The mortalities values were obtained by probit analysis. One potential ratio was selected for efficacy study among all ratios. The 2:8 ratio was found effective against the larvae of Cx. quinquefasciatus and An. stephensi. Whereas, a 4:6 ratio was found effective against the larvae of Ae. aegypti. The larvae of Cx. quinquefasciatus were found highly susceptible to the purified metabolites of A. niger than the larvae of An. stephensi and Ae. aegypti. Conclusion: The metabolites of A. niger may be environmental safer and a biocontrol agents for mosquitoes population in tropical countries where these vectors are significant.

Purification and characterization of an extracellular 24 kDa chitobiosidase from the mycoparasitic fungus Trichoderma saturnisporum

A Trichoderma saturnisporum Hamill isolate GITX-Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek-Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE-Cellulose anion-exchange and Sephacryl S-200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4-methylumbelliferyl -N,N ' diacetyl-β-D-chitobioside (4-Mu-(GluNAc)(2) , the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn(2+) and Zn(2+) stimulated the enzyme activity by 63% and 41%, respectively. The K(m) and V(max) values of the purified enzyme for 4-Mu-(GluNAc)(2) were 338.9 μM ml(-1) and 0.119 μM ml(-1) min(-1) , respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum.

Phosphoribosylamidotransferase, the first enzyme for purine de novo synthesis, is required for conidiation in the sclerotial mycoparasite Coniothyrium minitans

Coniothyrium minitans is an important sclerotial parasite of the fungal phytopathogen, Sclerotinia sclerotiorum. Previously, we constructed a T-DNA insertional library, and screened for many conidiation-deficient mutants from this library. Here, we report a T-DNA insertional mutant ZS-1T21882 that completely lost conidiation. In mutant ZS-1T21882, the T-DNA was integrated into a gene (CmPrat-1) which encodes phosphoribosylamidotransferase (PRAT, EC 2.4.2.14), an enzyme catalyzing the first committed step in de novo purine nucleotide synthesis. Gene replacement and complementation experiments confirmed that phosphoribosylamidotransferase is essential for conidiation of C. minitans. Mutant ZS-1T21882 did not grow on modified Czapek-Dox broth (MCD), but it grew well on MCD amended with IMP or AMP. The conidial production of this mutant was dependent on the dosage of IMP amended. At low concentrations, such as 0.1mM and 0.25mM, the mutant produced very few pycnidia, while up to 0.75mM or higher, the conidiation of this mutant was restored completely. cAMP could not restore the conidiation of mutant ZS-1T21882 when amended into MCD, but could when amended into PDA. Neither GMP nor cGMP could restore the conidiation in MCD or in PDA. Our findings suggest that phosphoribosylamidotransferase is essential for conidiation of C. minitans via adenosine related molecules. Furthermore, when dual cultured with its host, this mutant produced conidia in the host mycelium and on the sclerotia of S. sclerotiorum, but not in dead mycelium or on dead sclerotia, suggesting that C. minitans is likely to able to obtain adenosine or related components from its host during parasitization.

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.

The influence of different nitrogen and carbon sources on mycotoxin production in Alternaria alternata

The aim of this study was to determine the influence of different carbon and nitrogen sources on the production of the mycotoxins alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by Alternaria alternata at 28°C using a semi-synthetic medium (modified Czapek-Dox broth) supplemented with nitrogen and carbon sources. Additionally the effect of shaken and static cultivation on mycotoxin production was tested. Initial experiments showed a clear dependency between nitrogen depletion and mycotoxin production. To assess whether nitrogen limitation in general or the type of nitrogen source triggers the production, various nitrogen sources including several ammonium/nitrate salts and amino acids were tested. In static culture the production of AOH/AME can be enhanced greatly with phenylalanine whereas some nitrogen sources seem to inhibit the AOH/AME production completely. TA was not significantly affected by the choice of nitrogen source. In shaken culture the overall production of all mycotoxins was lower compared to static cultivation. Furthermore tests with a wide variety of carbon sources including monosaccharides, disaccharides, complex saccharides such as starch as well as glycerol and acetate were performed. In shaken culture AOH was produced when glucose, fructose, sucrose, acetate or mixtures of glucose/sucrose and glucose/acetate were used as carbon sources. AME production was not detected. The use of sodium acetate resulted in the highest AOH production. In static culture AOH production was also stimulated by acetate and the amount is comparable to shaken conditions. Under static conditions production of AOH was lower except when cultivated with acetate. In static cultivation 9 of 14 tested carbon sources induced mycotoxin production compared to 4 in shaken culture. This is the first study which analyses the influence of carbon and nitrogen sources in a semi-synthetic medium and assesses the effects of culture conditions on mycotoxin production by A. alternata.

Detoxification of malachite green and textile industrial effluent by Penicillium ochrochloron

Malachite green was detoxified into p-benzyl-N,N-dimethylaniline and N,N-dimethyl-aniline hydrochloride by Penicillium ochrochloron. Degradation metabolites were analyzed by TLC, HPLC, and FTIR and identified by GCMS analysis. Phytotoxicity testing revealed the nontoxic nature of these metabolites. The percentage decolorization of malachite green (50 mg/L) was 93% in czapek dox broth after 14 h with an optimum pH of 7 at 30°C. The induction in the activity of lignin peroxidase after degradation suggested that the degradation of malachite green was peroxidase-mediated. Fungal culture was also found to have detoxified the textile effluent. The values of TDS, TSS, COD, and BOD were reduced in the treated samples compared to the control effluent. The treated effluent was non-toxic to the plants of Triticum aestivum and Ervum lens Linn, and the amount of total chlorophyll was higher in plants with treated effluent when compared to control effluent.

Pathogenicity of Fusarium oxysporum against the larvae of Culex quinquefasciatus (Say) and Anopheles stephensi (Liston) in laboratory

The entomopathogenic fungi Fusarium oxysporum are the next generation mosquito controlling agent. F. oxysporum basically contains unique toxin and can be a selectively good agent in tropical countries. We are reporting here the efficacy of the metabolites of F. oxysporum against the larvae of Anopheles stephensi and Culex quinquefasciatus in the laboratory. F. oxysporum was grown on Czapek Dox broth. The bioassays were run at five different concentrations (1.30, 1.60, 1.77, 1.90, and 2.00 ppm). The LC(50), LC(90), and LC(99) values with 95% fiducial limits and probit equations were calculated by probit analysis. The mortality was observed after 24, 48, and 72 h against all instars. The LC(90) values in the case of C. quinquefasciatus after 48 h when calculated were 1.85, 1.92, 1.87, and 1.87 ppm, respectively, while LC(99) values calculated were 2.24, 2.25, 2.18, and 2.19 ppm. Moreover, after 48 h in the case of A. stephensi, the LC(50) values for the first, second, third, and fourth instars were recorded as 1.48, 1.51, 1.71, and 1.50 ppm, respectively. The LC(90) values recorded were 1.88, 1.91, 1.93, and 1.89 ppm and LC(99) values observed were 2.36, 2.23, 2.26, and 2.21 ppm. The results obtained 24, 48, and 72 h have been compared and it was observed significantly that 48 h after exposure the metabolite has more pathogenicity. The results of the metabolites of F. oxysporum may be considered as a new bio-control agent for vector mosquitoes if the field trial succeeds.

Formation of exudate droplets by Metarhizium anisopliae and the presence of destruxins

Nutritional conditions causing droplet exudation by Metarhizium anisopliae var. anisopliae were studied. Exudation in droplets occurred only on media with more than one carbon source and was highly dependent on the ratio of a well metabolized sugar such as trehalose and a nonpreferred sugar, in particular arabinose. Exuded droplets contained destruxin A, B and E in concentrations similar to those on submerged culture on Czapek Dox medium with equivalent C : N ratios but was clearly less than previously reported on standard Czapek Dox or Sabouraud dextrose broth. Destruxins also were found in agar samples from directly below mycelium and from up to 2 cm from the colony edge. Exudates retrieved from different media were proven to have Pr1 protease-related enzyme activity. Additional HPLC analysis indicated that droplets from diverse media did not differ in their sugar and acid content. A hypothesis is presented regarding the trigger for guttation in Metarhizium during growth under these conditions.

Characterization and Antimicrobial Activities of a Metabolite from a New <i>Streptomyces</i> Species from Bangladeshi Soil

A new actinomycete strain was isolated from Western part of Bangladesh and identified as a new Streptomyces species on the basis of its morphological, biochemical, cultural characteristics and 16S rRNA data. The present paper describes the isolation and characterization of compound 1 from this new Streptomyces species with the help of various chemical and spectroscopic methods. Antimicrobial activity of compound 1 was tested by disc diffusion assay method and compared with that of standard antibiotics (Kanamycin for antibacterial activity and Nystatin for antifungal activity). The compound has been found to exhibit moderate to strong antimicrobial activity against the test organisms. Cytotoxicity of the compound 1 and the pet. ether extract of Czapek Dox (alkaline) broth of Streptomyces species was evaluated in brine shrimp bioassay with LC50 values of 23.85 µg/ml (ppm) and 19.95 µg/ml (ppm), respectively. Keywords: Streptomyces; Antimicrobial activity; Cytotoxicity. © 2010 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. DOI: 10.3329/jsr.v2i1.3079 J. Sci. Res. 2 (1), 178-185 (2010)

Optimization of extracellular lipase production in Colletotrichum gloeosporioides by solid state fermentation

Twenty two endophytic fungi isolated from oil seeds were screened for their ability to produce extracellular lipolytic enzymes in solid state fermentation. Among them, the most productive strain, identified as Colletotrichum gloeosporioides was cultivated for lipase production in solid state fermentation (SSF) using oil-mill residue as a solid support. The elaboration of extracellular lipases from C. gloeosporioides on different residual cheap oil substrates was tested. Among these, pongamia oil cake (POC) showed maximum lipase activity (983 U/g dry matter (DM)) followed by coconut oil cake (COC) (925 U/g DM). These two oil industrial residues were chosen for enhanced lipase production. The maximum enzyme yield was obtained when POC was impregnated with CDB (Czapek-Dox Broth) in the ratio of 1:1.5 and with (906 U/g DM). Triton X 100 as the extractant from the fermented solids yielded 1020 U/g DM. The optimum temperature for lipase secretion by C. gloeosporioides was found to be 25°C. Xylose as the carbon source and peptone as the nitrogen source were favorable for the secretion of the enzymes. Tween 60 served as a good lipid substrate for lipase production (1170U/g DM) when POC was the substrate. Amending the POC with Magnesium sulphate as a metallic ion source induced good lipase activity (1240 U/g DM) by C. gloeosporioides. The Sunflower oil was found to be best co-vegetable oil substrate to induce the enzyme (2560 U/g DM). Triton X100 served as the surfactant for lipase secretion in C. gloeosporioides. Thus, pongamia oil cake can be used as cheap substrate for enhanced extracellular lipase production by C. gloeosporioides .

Optimization of Extracellular Lipase Production in Colletotrichum Gloeosporioides by Solid State Fermentation

Twenty two endophytic fungi isolated from oil seeds were screened for their ability to produce extracellular lipolytic enzymes in solid state fermentation. Among them, the most productive strain, identified as Colletotrichum gloeosporioides was cultivated for lipase production in solid state fermentation (SSF) using oil-mill residue as a solid support. The elaboration of extracellular lipases from C. gloeosporioides on different residual cheap oil substrates was tested. Among these, pongamia oil cake (POC) showed maximum lipase activity (983 U/g dry matter (DM)) followed by coconut oil cake (COC) (925 U/g DM). These two oil industrial residues were chosen for enhanced lipase production. The maximum enzyme yield was obtained when POC was impregnated with CDB (Czapek-Dox Broth) in the ratio of 1:1.5 and with (906 U/g DM). Triton X 100 as the extractant from the fermented solids yielded 1020 U/g DM. The optimum temperature for lipase secretion by C. gloeosporioides was found to be 25°C. Xylose as the carbon source and peptone as the nitrogen source were favorable for the secretion of the enzymes. Tween 60 served as a good lipid substrate for lipase production (1170U/g DM) when POC was the substrate. Amending the POC with Magnesium sulphate as a metallic ion source induced good lipase activity (1240 U/g DM) by C. gloeosporioides. The Sunflower oil was found to be best co-vegetable oil substrate to induce the enzyme (2560 U/g DM). Triton X100 served as the surfactant for lipase secretion in C. gloeosporioides. Thus, pongamia oil cake can be used as cheap substrate for enhanced extracellular lipase production by C. gloeosporioides .

액체와 고체 발효 조건에서 Aspergillus niger의 셀루로오스계 효소 생산

Aspergillus niger가 액체발효(SF) 와 고체발효의 시험 규모 조건에서의 셀루로오스계 효소의 생산을 비교하였다. 액체배지는 0.5% 셀루로오스가 함유된 Czapek Dox를 사용하였고, 고체 지지체로 사용한 쌀겨는 셀루로오스로 적시고 SSF 발효를 위하여 Czapek Dox 배지를 첨가하였다. CMCase, 여지 paperase 그리고 <TEX>${\beta}$</TEX>-Glucosidase 의 생산을 경시적으로 측정하였다. SSF 배양에서의 3일간의 효소 생산량은 SF 에서의 7일간 배양과 같았다. 따라서 SSF 조건이 SF 배양 조건보다 많은 효소를 생산함을 알 수 있었다. SSF 조건에서 FPase, CMCase 및 <TEX>${\beta}$</TEX>-glucosidase의 최대 활성은 0.40, 0.62 및 0.013 U/ml 였으나, SF 조건에서는 0.13, 0.06 및 0.0013 U/ml의 활성을 나타내었다. 결론적으로 Aspergillus niger에 의해 생산되는 셀루로오스계 효소의 생산을 위해서는 SSF 발효 조건의 선택이 유리하다는 것을 알 수 있었다. Microbial production of cellulolytic enzymes by Aspergillus niger in solid state fermentation (SSF) and submerged state fermentation (SF) in laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used for cultivation in SF, whereas rice bran was used as a solid support, moistened with cellulose, amended Czapek Dox broth for growth in SSF. The production of Carboxymethyl cellulase, Filter paperase and <TEX>${\beta}$</TEX>-Glucosidase was monitored at regular intervals. The peak production of the enzymes occurred within 3 days of incubation in SSF as against <TEX>$\geq$</TEX> 7 days in SF. SSF gave higher yields of enzymes in comparison to SF. Maximum titres of 0.40, 0.62 and 0.013 U/ml in respect of FPase, CMCase and <TEX>${\beta}$</TEX>-glucosidase in SSF were recovered as against 0.13, 0.06 and 0.0013 U/ml in SF respectively, at their respective peak time intervals. Hence, SSF appeared to be a better choice for production of cellulolytic enzymes by Aspergillus niger.

Mycotoxigenic Potential of Ten Fusarium species Grown on Sorghum and in vitro

The objective of this study was to determine the mycotoxigenic potential of 12 Fusarium isolates (10 species), including six isolates (4 species) from sorghum. The species were: F. thapsinum, F. semitectum, F. proliferatum and F. chlamydosporum isolated from molded sorghum seed; F. poae, F. graminearum and F. sporotrichioides from barley seed with Fusarium head blight; F. acuminatum from wheat seed; F. verticillioides from infected corn seed; and F. nygamai isolated from soil. Fumonisin and zearalenone concentrations were measured following incubation on autoclaved sorghum seed for 21 days at 25°C, while fusaric acid was measured in mycelia harvested from Czapek Dox broth cultures. F. thapsinum (SC8 and CS121) and F. semitectum (SC7) produced fusaric acid only (4.59-64.13 mg g-1). F. graminearum (KB172) and F. semitectum (CS152) produced zearalenone only (73.4 and 799.3 μg g-1, respectively). F. proliferatum (CS183), F. verticillioides (TX02) and F. nygamai produced both fumonisin (1.92-6.05 μg g-1) and fusaric acid (39.4-234.17 mg g-1). F. poae (KB652), F. acuminatum (Ark), F. chlamydosporum (CS102) and F. sporotrichioides (KB662) did not produce any of these three mycotoxins. Five of the six Fusarium isolates (three species) isolated from sorghum had mycotoxigenic potential. Fusarium spp. naturally occurring on sorghum in the field have the potential to contribute to mycotoxin contamination, either singly or in combination

Production of phytotoxic metabolites by five species of Botryosphaeriaceae causing decline on grapevines, with special interest in the species Neofusicoccum luteum and N. parvum

In recent years an increasing number of species of Botryosphaeriaceae have been associated with grapevine decline worldwide. Five species isolated from declining grapevines in Spain (Botryosphaeria dothidea, Diplodia seriata, Dothiorella viticola, Neofusicoccum luteum and N. parvum) were checked for toxin production in liquid cultures. Cultural conditions for all fungi were adjusted to obtain optimal production of phytotoxic culture filtrates, by growing the fungi in steady liquid cultures of Czapek–Dox broth for different time intervals. Phytotoxicity of D. seriata and N. parvum reached a maximum after 14 days while the remaining species showed the highest phytotoxicity levels after 21 days in culture. All fungi produced hydrophilic high-molecular weight compounds with phytotoxic properties. In addition, N. luteum and N. parvum produced lipophilic low-molecular weight phytotoxins, not detected consistently among the remaining species. This led to a more exhaustive study on the phytotoxicity of N. luteum and N. parvum. Culture filtrates and corresponding extracts of both species were consistently highly phytotoxic in different assays. The gas-chromatography analysis of the acetylated O-methyl glycosides of the phytotoxic exopolysaccharides produced by N. parvum showed these substances to be composed mainly of glucose, mannose and galactose. Results suggest that phytotoxic metabolites could be involved in the virulence of both species in planta.

Trichoderma harzianum and its Metabolite 6‐Pentyl‐alpha‐pyrone Suppress Fusaric Acid Produced by Fusarium moniliforme

AbstractThe interaction of the pathogen Fusarium moniliforme and two antagonistic Trichoderma harzianum isolates was studied especially with respect to their secondary metabolites fusaric acid (FA) and 6‐pentyl‐alpha‐pyrone (6PAP). Among 10 isolates of F. moniliforme screened for FA production on maize kernels, the isolate 8 accumulated the highest amount of FA (678 μg/g). Mycelial growth and production of FA by isolate 8, determined in different liquid media revealed that the highest biomass and FA were produced in Czapek Dox Broth (CDB) followed by Richard’s solution. The amount of FA per gram mycelial dry weight reached its maximum in CDB and Richard’s solution after 14 days of incubation. Mycelial growth and conidia production of both Trichoderma isolates (T16 and T23) were retarded by increasing concentrations of FA in agar medium. At FA concentration of 300 mg/ml the radial mycelial growth of the isolates T16 and T23 were retarded by 32.5% and 45%, respectively. Conidia production was diminished in a similar extent as mycelial growth. Both T. harzianum isolates were capable to degrade FA in potato dextrose broth medium, particularly when lower doses of FA were present. In the presence of 50 mg/ml FA in the culture medium, the isolates T23 and T16 reduced FA by 51.4% and 88.4%, respectively, 9 days post‐inoculation. The antifungal metabolite 6PAP, isolated from T. harzianum T23 cultures, was introduced at different concentrations into 2‐day‐old cultures of F. moniliforme. After further 5 days of incubation of F. moniliforme in the presence of 6PAP, the FA contents per gram mycelial dry weight were significantly decreased compared to control cultures where 6PAP was absent. Dosages of 300 and 400 mg/l of 6PAP in the cultures retarded FA accumulations by 62.5% and 77.2%, respectively. The current results, however, provided the first evidence for activity of 6PAP, as a Trichoderma secondary metabolite, on degrading/synthesis suppression of the Fusarium toxin FA.

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.

Inhibition of Aflatoxin Production of Aspergillus flavus by Lactobacillus casei

Lactobacillus casei KC-324 was tested for its ability to inhibit aflatoxin production and mycelial growth of Aspergillus flavus ATCC 15517 in liquid culture. Aflatoxin B1 biosynthesis and mycelial growth were inhibited in both simultaneous culture and individual antagonism assays, suggesting that the inhibitory activity was due to extracellular metabolites produced in cell-free supernatant fluids of the cultured broth of L. casei KC-324. In cell-free supernatant fluids of all media tested, deMan, Rogosa and Sharpe broth, potato dextrose broth, and Czapek-Dox broth +1% yeast extract showed higher anti-aflatoxigenie activity. In these ease, fungal growths, however, was not affected as measured by mycelial dry weight. The anti-aflatoxigenie metabolites from L. casei KC-324 were produced over wide range of temperatures between 25°C and 3TC. However, these metabolites were not thermostable since the inhibitory activity of the supernatant was inactivated within 30 minutes at 100°C and 121°C. The inhibitory activity was not influenced by changing pH of supernatant between 4 and 10. However, the antiaflatoxigenic activity was slightly reduced at pH 10.