Bioassay of Mildiomycin and a Rapid, Cost-Effective Agar Plug Method for Screening High-yielding Mutants of Mildiomycin

Summary A simple, reliable and low-cost agar diffusion bioassay for quantitative determination of mildiomycin was developed using a strain of Rhodotorula rubra AS 2.166 as the indicator organism and potato dextrose agar at pH 7.0 as the test medium. With equivalent precision and accuracy to HPLC analysis, this method was applied to analyse mildiomycin in complex culture broth during the fermentation process. A modified agar plug method based on the bioassay was constructed for rapid and efficient screening of high-yielding mutants of mildiomycin. Within four weeks, a high production strain, the mildiomycin productivity of which was 75.5% higher than the parent strain, was obtained from 15,000 mutants.     

Studies on the fungal infestation of five traditionally smoke-dried freshwater fish in Ago-Iwoye,Nigeria

Samples of traditionally smoke-dried Clarias gariepinus (Burchell), Chrysichthys nigrodigitatus (Lacepede), Sarotherodon galilaeus (Trewavas), Heterotis niloticus (Cuvier) and Heterobranchus bidorsalis (Geoffroy) were obtained from Oja Oba Market, Ago-Iwoye, Nigeria and examined for fungal infestation. The fish samples incubated on potato dextrose agar (PDA) for 7 days showed fungal infestation. Fungi isolated and identified included Mucor sp., Aspergillus sp., Rhizopus sp. and Fusarium sp. Six fungal species were isolated from C. nigrodigitatus, five each on C. gariepinus and H. niloticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of coal solubilization by bacteria and fungi

Coal-solubilizing agents produced byTrametes versicolor, Phanerochaete chrysosporium, Aspergillus sp., a bacterial consortium, and a bacterial isolate,Arthrobacter sp., from that consortium were compared in terms of pH dependence, thermostability, molecular mass, mechanism of action, and product diversity. The thermostability and low molecular weights exhibited by the coal-solubilizing agents indicated a non-enzymatic mechanism of action. Competition studies using cupric copper indicated that coal solubilization by these agents involved metal chelation. Results demonstrated that oxalate could account for some but not all of the coal solubilization observed forT.versicolor andP.chrysosporium. The very low levels of oxalate detected inAspergillus sp. and the bacterial cultures indicated that oxalate is not an important factor in coal solubilization by these microbes. When subjected to gel permeation chromatography, the soluble coal products generated by each microbial coal-solubilizing agent yielded unique molecular mass profiles suggesting substantial product diversity. Such diversity increases the possibility of identifying potentially valuable compounds and extending the commercial utilization of coal.Abbreviations A₄₅₀, A₂₆₀ absorbances respectively at 450 nm and 260 nm - CSA coal-solubilizing agent - CSU coal-solubilizing unit - GPC gel permeation chromatography - MEA malt extract agar - PDA potato dextrose agar - SDA Sabouraud dextrose agar - SDB Sabouraud dextrose broth - SEM standard error of the mean - Tris tris(hydroxymethyl)aminomethane - TSA trypticase soy agar - TSB trypticase soy broth     

A method for both mass and individual rearing of fungivorous astigmatid mites (Acari)

Several species of common fungi were assessed as food for fungivorous astigmatid mites. Hypocrea nigricans, Botrytis cinerea and Flammulina velutipes were generally good food sources for most mites examined. Fungal mycelia growing on PDA (potato dextrose agar) medium were not only nutritionally adequate but the system also maintained high humidity through the water-based agar medium. Among acarid mites, most species of Rhizoglyphinae could be reared easily with the method. Although filter-feeding histiostomatid mites do not feed directly on hyphae, some species were successfully maintained with the same method through multiple generations. Presumably, these mites obtained sufficient nutrition from the agar medium and fungal metabolites leaching into it. Most species ultimately produced dispersing heteromorphic deutonymphs on these media. Individual mites were also maintained in isolation within glass rings on fungal colonies. Using this technique, we were able to compare developmental periods, fecundity and survival periods of mites reared under different conditions.     

Aberrant Growth and Conidiation in Wild-Type Cultures of Fusarium Species from Wheat

Fusarium avenaceum, F. graminearum, F. poae and F. tricinctum showed abnormal growth, morphology and conidiation, and a tendency to produce crystals, inclusion bodies and sclerotia when freshly isolated from wheat stem bases or kernels onto low‐carbon potato dextrose agar (PDA). Observations of alterations in conidiation and conidium morphology are particularly significant, as these are the principal morphological diagnostic characteristics for Fusarium species. The fungi had normal growth when sub‐cultured onto standard PDA, suggesting that a balance of nutrients was responsible for the effects. Specific causes are discussed in detail in relation to published information. The importance of standard media in the identification of Fusarium species is emphasized, whilst non‐standard media may be useful for specific purposes, including routine isolation of fungi from mixed communities of species with different nutrient requirements.     

A Method for Purifying Cochliobolus sativus Cultures Contaminated with Bacteria

A simple method was developed for purifying Cochliobolus sativus cultures contaminated with bacteria. In this method the contaminated fungal culture is scraped from the surface of the potato dextrose agar (PDA) growth medium. Small leaf pieces (approximately 0.5 cm) of a highly susceptible genotype of barley were placed on the scraped surface of PDA and incubated for 72 h. Disease symptoms of C. sativus were detected on the leaf pieces. Colonies that developed from these pieces on new PDA medium were free from bacteria.     

Yeast diversity in a mesotrophic lake on the karstic plateau of Lagoa Santa,MG-Brazil

The yeast diversity in a paleo-karstic lake of the Lagoa Santa plateau was studied during March 1986–March 1987. Water samples were collected monthly at five stations and the yeasts were isolated at 25 °C on Sabourad dextrose agar supplemented with 0.5% of yeast extract and 10 mg% of chloramphenicol. August and February showed the highest numbers of isolates, coinciding, respectively with the beginning and the ending of stratification. Of 56 isolated species, Aureobasidium pullulans, Candida famata, Cryptococcus albidus, Cryptococcus laurentii, Rhodotorula glutinis, Rhodotorula rubra and Trichosporon cutaneum occurred at the highest frequencies. A. pullulans showed greatest prevalence during the dry period (winter), whereas C. famata, Cr. albidus, Cr. laurentii and Rh. rubra were prevalent during the rainy season (summer). Tr. cutaneum was distributed between July and November. Most isolates yeasts are associated with plants and soils, and are important in decomposition of aquatic macrophytes. Furthermore, a possible role of these species as indicators of pollution is discussed.     

Removing of Crude Oil From Polluted Areas Using the Isolated Fungi From Tehran Oil Refinery

The pollution of soil and the subsurface environment by crude oil spill and petroleum products spill is a major concern around the world. The aim of this research was to investigate the ability of fungi isolated from Tehran oil refinery area in removing crude oil and to evaluate their enzymatic activities. Plant root samples were collected from the polluted and control areas, and rhizospheral fungi were isolated and determined using the laboratory methods and taxonomic keys. Seven fungal species were isolated and then cultured in potato dextrose agar (PDA) media containing 0–15% (v/v) crude oil. Oil removal was determined after a one-month growth of fungal colonies and then compared with the control media. The results showed that the studied fungi were able to remove crude oil from the media. The highest removal efficiency was observed in Aspergillus sp. Total protein content and enzymatic activity (of peroxidase and catalase) increased with increasing crude oil pollution. The highest enzymatic activity was evaluated in Aspergillus sp. growing in media containing 15% petroleum and the lowest activity was found in non-polluted groups. Results showed that there is a direct correlation between oil-removing potency and enzymatic activity. Aspergillus sp. showed the highest enzyme activity and also the highest petroleum removal efficiency.     

Gari as culture medium for moulds

No significant differences (P<0.05 occurred in the frequency of isolation from soil of four common moulds – Rhizopus stolonifer, Monilia sitophila, Aspergillus niger and Penicillium sp., on malt extract agar (MEA) and gelled gari (carbohydrate gel). Growth of the moulds on slide microcultures of potato dextrose agar (PDA) and gari showed typical diagnostic features of the organisms, which were often clearer on gari. It is concluded that gari, which is much cheaper and far more readily available in Nigeria than MEA or PDA, is an effective alternative to the two standard mycological media.     

Effect of spore concentration on germination and autotropism inTrichoderma hamatum

Summary The effect of spore concentration on spore germination and germtube growth ofTrichoderma hamatum on water agar and on potato dextrose agar (PDA) was studied. Increasing inoculum size up to 10⁹ spores/plate on PDA and up to 10⁷ spores/plate on water agar shortened the incubation period required for germtubes emergence and increased germination rate. However, on water agar germination was inhibited at 10⁸ and was completely arrested at 10⁹ spores/plate. Inhibition in germination of 10⁷ spores/plate was observed on water agar when the plates were preincubated with 10⁹ spores/plate for 5 h or more. Addition of glucose and ammonium nitrate to the water agar medium allowed only 25% of the spores to germinate at 10⁹ as compared to 78% at 10⁷ spores/plate after 8 h of incubation. Addition of polysaccharides to the C+N supplemented medium, significantly increased germination up to 84% as compared to 100% on PDA, after 8 h of incubation. Germlings ofTrichoderma hamatum phialospores exhibited positive autotropism and anastamosis on both media. The phenomenon was positively related to inoculum size, being most pronounced at 10⁷ spores/plate.     

Characterization of Sporulation of Alternaria alternata f. sp. sphenocleae

Studies were conducted on agar media to characterize the factors for the optimization of sporulation of Alternaria alternata f. sp. sphenocleae , a fungal pathogen being evaluated as a biological control agent for Sphenoclea zeylanica (gooseweed). A. alternata f. sp. sphenocleae conidiation was affected by nutrition, temperature, light conditions, and moisture. On all agar media tested, except for half-strength potato dextrose agar (&#189; PDA) and V-8 juice agar (VJA), exposure to different light conditions did not have any significant effect on conidia production. However, when comparing &#189; PDA and VJA, sporulation under constant near-ultraviolet (NUV) light at 28 o C increased markedly on VJA, but decreased substantially on &#189; PDA. This trend, however, was opposite under dark conditions since &#189; PDA produced the greatest number of conidia whereas a 75% reduction in conidia production occurred on VJA in the dark. On all the standard agar media evaluated, the most virulent conidia were obtained on &#189; PDA at 28 o C under constant NUV incubated for 4 weeks. Sporulation of A. alternata f. sp. sphenocleae using the sporulation medium (S-medium) technique was rapid. Conidia were produced within 24 h and continuous sporulation was still observed until 120 h. The best primary agar media for conidia production were PDA, &#189; PDA and VJA, while water agar was the poorest. Conidia production was optimized with the addition of 20 g l -1 of calcium carbonate (CaCO 3 ) and the addition of 2 ml of sterile distilled water on the medium. The most virulent conidia were produced when the primary agar was &#189; PDA, the CaCO 3 concentration was 20 g l -1 , and the cultures were incubated at 18 o C in the dark. Conidiophore induction occurred on nutrient rich media and was stimulated by NUV, while formation of conidia proceeded in darkness after nutrients were depleted under warm dry or cool moist conditions. Culture media, growth conditions, and CaCO 3 affected the inoculum potential of A. alternata f. sp. sphenocleae conidia.     

Comparison ofRhizoctonia solani isolates from web blight and basal canker of cowpea and from soil

Summary Isolates ofRhizoctonia solani from web blight and stem basal canker of cowpea and those obtained from soil had similar linear growth rates on potato dextrose agar (PDA) at various temperatures but differed in other features. The web blight isolates differed from the basal canker and the soil isolates in cultural appearance on PDA and on potato marmite agar (PMA). The web blight isolates readily formed discrete sclerotia on PDA, PMA and soil but the other isolates did not. In greenhouse tests, the former were generally the most virulent in inciting foliage and stem basal necrosis and damping-off of seven crop species. Of the plants tested, the legumes were the most susceptible toR. solani.     

A Comparative Study of Caffeine Degradation by Four Different Fungi

The objective of the present study is to investigate the caffeine-degrading abilities of different fungi and to apply this knowledge to environmental remediation and industrial decaffeination process. Chrysosporium keratinophilum, Gliocladium roseum, Fusarium solani, and Aspergillus restrictus were isolated from the coffee pulp obtained from a coffee estate. Pure cultures of fungi were isolated on standard conventional potato dextrose broth (PDB) medium and authenticated. Pure cultures were subjected to a caffeine tolerance study at different concentrations of caffeine (1–8 g/L) in potato dextrose agar (PDA) and minimal media. On PDA, Fusarium solani could tolerate caffeine concentration up to 8 g/L, whereas Chrysosporium keratinophilum, Gliocladium roseum, and Aspergillus restrictus could tolerate up to 6 g/L. On minimal agar medium containing different concentrations of caffeine (1–8 g/L), Fusarium solani tolerated up to 8 g/L and the other fungi up to 2 g/L. A time-bound caffeine degradation study was undertaken at 1 g/L concentration of caffeine and glucose in nitrogen-containing and nitrogen-free liquid minimal media by subjecting the four fungi to shake flask culture at 120 rpm and 30°C. Degradation of caffeine up to 7 days at 24-h intervals was analyzed by high-performance liquid chromatography (HPLC). Gliocladium roseum followed by Aspergillus restrictus showed maximum degradation of caffeine at 0.47 and 0.3 mg/ml, respectively, by 96 h in nitrogen-containing minimal medium, whereas Fusarium solani showed maximum degradation of caffeine by 48 h (0.35 mg/ml) and Chrysosporium keratinophilum by 72 h (0.29 g/ml). In nitrogen-free minimal medium, Chrysosporium keratinophilum showed maximum degradation of caffeine at 72 h (0.45 mg/ml), followed by Gliocladium roseum, Fusarium solani (0.3 mg/ml), and Aspergillus restrictus (0.25 mg/ml) at 96 h. Overall, Chrysosporium keratinophilum showed a comparatively higher rate of caffeine degradation in minimal medium with or without a nitrogen source as compared with the other three fungi, indicating that nitrogen affects caffeine metabolism.     

Cultural, morphological, pathogenic and molecular variability amongst tomato isolates of Alternaria solani in India

Early blight (Alternaria solani) is an important disease causing severe damage in tomato. The eleven isolates of A. solani designated as So, Dh, Sh, Va-5, Ka, Ma, Hy, Ba-1, My, Va-3 and Mi were collected from different agroclimatic conditions and these isolates were characterized for cultural, morphological, pathogenic and molecular variations. The pigmentation varied from yellow, brown, black, brownish to greenish black in isolates of A. solani on potato dextrose agar medium. In general, radial growth of all isolates ranged between 14.9 mm and 32.2 mm on PDA and 24.3 mm to 53.7 mm on three selective media i.e., ASM, V-8 juice agar and V-8 juice agar (synthetic) on the fourth day. The fastest radial growth was recorded in the So isolate and slowest in the Ka isolate on PDA, while isolates Dh, Ba-1 and Va-3 were recorded to be faster in growth on ASM, V-8 juice agar and V-8 juice agar (synthetic) medium. The thickness of conidiogenous hyphae varied between 1.17 μ and 9.56 μ, with maximum in the Va-5 and Ma isolates. Most of the isolates showed smooth mycelial growth with circular and irregular margin and without concentric zonation. Sporulation was not found in any of the isolates on four different nutrient media, whereas conidiogenous hyphal length was observed in V-8 juice agar medium only. Based on the pathogenicity, isolates of A. solani were rated as virulent or less virulent based on percentage disease incidence data. Molecular variability studies were also done to find out the best annealing temperature and eighty-six primers were screened to select for maximum polymorphism of DNA. The best annealing temperature was recorded between 32.5 °C and 34.0 °C for the pathogen, and most efficient amplification and polymorphism of DNA was found with random primer 5′-CGCGTTCCTG-3′.     

Mycelial compatibility in Valsa malicola,the causal agent of canker disease in Iran

Mycelial compatibility of 62 isolates of Valsa malicola from different hosts and areas of Iran were investigated on potato dextrose agar (PDA) and oat meal agar (OMA). Four mycelial compatibility groups (MCGs) on PDA, 1–4, including three single membered (1–3) and a 58 membered (4) were identified. However, 8 MCGs, 1–8, consisting of 6 single membered (1–3, 5, 6 and 8), a 6 membered (4) and a 50 membered (7) were identified on OMA. On PDA, the number of groups and the time to achieve results were less than on OMA as well as the barrage zones were clearer on PDA than OMA. There was no correlation between groups and host or geographical origins of the isolates. The low number of identified MCGs on both culture media revealed low genetic diversity of investigated isolates of V. malicola.     

Time-Dependent Sporulation, Conidial Size and Germ Tube Formation in Alternaria tenuissima (Kunze ex Pers.) Wiltshire on Different Media

Time-dependent sporulation, conidial size and germ tube formation in Alternaria tenuissima (Kunze ex Pers.) Wiltshire on different media were studied. The results indicate that maximum number of first spore was formed on potato dextrose agar (PDA) and new leaf extract of pigeon pea followed by old leaf extract of pigeon pea and distilled water (DW). The sporulation was relatively poor on other media. The maximum number of spores in a chain were produced on PDA and albumen +0.9% sodium citrate. Only one spore was seen in DW. According to time-dependent sporulation, the media were divided into three categories: on some media the first spore was formed in 18 h, in another group it took 30–36 h and in the third group the first conidium was formed after 40 h. Four germ tubes were commonly formed on most of the media. The largest conidia were observed on potato dextrose and the smallest ones were seen on DW.     

Fungal colonization of computer diskettes and other magnetic media

Computer diskettes can be colonized by saprophytic fungi, especially in the humid tropics. Fungi of the generaAlternaria, Aspergillus, Epicoccum, Paecilomyces, Penicillium, andTrichoderma were observed on diskettes from several tropical countries. Common saprophytic fungi from Minnesota colonized clean standard and high density diskettes in growth chambers, indicating that fungal contamination could occur wherever temperature and humidity were adequate.Fusarium species infested diskettes buried in garden soil in Minnesota. Audiotapes, videotapes, and computer magnetic tapes chemically resemble diskettes and also can be colonized by fungi, as can photographic film. The Mylar core of these magnetic media did not support the growth ofPenicillium glabrum, the most aggressive fungus in diskette inoculation studies. However, growth of several fungal species was enhanced when the common plasticizer, lecithin, was added in powdered form to nutrient agar, suggesting that this ingredient of the diskettes may be metabolized by the fungi.Abbreviations DD double density - HD high density - MA malt agar - NA nutrient agar - PDA potato dextrose agar - SEM scanning electron microscopy     

Growth and mycotoxin production by <Emphasis Type="Italic">Chaetomium globosum</Emphasis>

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.     

Fungi in the indoor environment of flour mill in Lucknow

Forty species of fungi were recovered from the indoor air of flour mills by exposing petri plates containing potato dextrose, czapek-dox, and sabouraud agar media for 5 minutes. A rotorod sampler was used. The fungal flora of the nearby outdoor environment was also studied for comparison. Species of genus Aspergillus dominated in the mills, being represented by 16 species including one ascosporic species. Other species were of genera Cladosporium and Fusarium. Variations in the fungal population in different months of the survey year were also observed.

Identifiable fungal spores recovered using the rotorod sampler were Alternaria (17% occurrence), Curvularia lunata (10.6%), C. letramera (10%), Cladosporium (19%), Drechslera sp. (9%), Epicoccum sp. (5%), Pithomyces sp. (3%), Nigrospora sp. (10.5%), Stemphylium sp. (4.5%) and Torula sp. (4.5%). Mycelial fragments and unidentifiable spores were also seen in abundance.

Varying allergic responses of patients tested intradermally for the antigens of six Aspergilli, viz., Aspergillus flavus, A. fumigatus, A. melleus, A. niger, A. niveus, and A. terreus were also recorded.     

Transmission of the mycoparasite Coniothyrium minitans by collembolan Folsomia Candida (Collembola: Entomobryidae) and glasshouse sciarid Bradysia sp. (Diptera: Sciaridae)

Folsomia Candida was maintained on potato dextrose agar (PDA) plates precolonised by the mycoparasite Coniothyrium minitans for 3 yr but the sciarid Bradysia sp. survived for a maximum of only three generations. Collembolans and sciarid larvae from these cultures were able to transmit C. minitans to uninoculated PDA plates through the survival of spores in faecal pellets. Adult and larval sciarids also transmitted C. minitans from PDA culture to uninoculated PDA plates by contamination on the cuticle. In soil and sand both sciarids and collembolans were able to transmit C. minitans from C. m/m'tans-inoculated to uninoculated sclerotia of Sclerotinia sclerotiorum. Inoculation of sclerotia with C. minitans enabled greater populations of larger collembolans to develop. In the glasshouse where C. minitans had been applied to the soil, one adult sciarid and four collembolans out of 70 and 101 insects collected respectively yielded C. minitans on placement onto PDA + Aureomycin.