Guttation droplets of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> and <Emphasis Type="Italic">Penicillium verrucosum</Emphasis> contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.     

In vitro conidial germination in Arthrinium aureum and Arthrinium phaeospermum

This paper describes the microscopic details of conidial germination and the influence of pH, sodium chloride concentration, 3% glucose, 3% saccharose and ultraviolet irradiation on the conidial germination in Arthrinium species. Under laboratory conditions, germination started after an incubation period of 90 minutes in 2% malt extract broth at 25 degrees C. In vitro, the conidia of Arthrinium species have a very low percentage of germination (A. phaeospermum: 7.9%; A. aureum: 15.8%). Conidia of this genus have a characteristic equatorial slit. Conidia may break spontaneously at this slit, releasing their cytoplasmic contents. Arthrinium phaeospermum attains its optimum germination percentage when its conidia are suspended in a sterile saline solution (pH 3.5) in a water bath at 20 degrees C for 15 minutes before being inoculated on 2% malt extract agar. Conidial suspensions of A. aureum may be held in the same conditions, but for 30 minutes.     

Micropropagation of curry leaf tree

Murraya koenigii (curry leaf tree) is cultivated for its aromatic leaves which are used as condiment. Nodal cuttings from mature curry leaf plants cultured in Woody plant basal medium (WPM) supplemented with 4.4 μM benzyladenine (BA) and 4.65 μM kinetin produced 12–30 multiple shoots per node by the eighth week of inoculation. The shoots easily rooted in vitro in woody plant medium contained naphthalene acetic acid 1.35 μM NAA. Ninety percent of the plants survived transfer to a hardening chamber and were transferred to the field after three months. In vitro-developed shoots were also rooted ex vitro by dipping in 2.46 μM indole-3-butyric acid for one minute. They were transplanted to sand in a hardening chamber with 70–80% relative humidity and a temperature of 28±2 °C. Eighty to ninety percent of the ex vitro-rooted plants survived and were transferred to the field after 3 months. This revised version was published online in June 2006 with corrections to the Cover Date.     

Failure to decontaminate Glomus clarum NT4 spores is due to spore wall-associated bacteria

 Exposure of spores of Glomus clarum NT4 to solutions of chloramine-T (2.5–10% w/v) for 10–120 min failed to fully decontaminate all spores. Scanning electron microscopy did not show the presence of contaminants on treated spores, but transmission electron microscopy revealed bacterial cells embedded within the outer spore wall layer. Bacteria that remained protected within the spore walls were detected only when the spores were placed on appropriate media. Nutrient agar and tryptic soy agar supported relatively high levels of contaminant growth and were regarded as good media for assessing contamination, whereas the detection of contaminant growth on water agar required prolonged incubation. Contamination and germination of G. clarum NT4 spores following decontamination treatments were dependent on spore age. Generally, lower concentrations of chloramine-T and shorter incubation periods were required to reduce contamination of freshly harvested spores than of mature spores. Exposure to 10% chloramine-T for 120 min was required to reduce the levels of contamination of mature spores to ≤10%. Unfortunately, spore germination was compromised by rigorous decontamination treatments, thus the success of any decontamination procedure should be evaluated prior to its routine use. Moreover, if the interpretation of experimental results rests on the assumption of true surface sterility of VAMF spores, we suggest that the axenic condition of spores be confirmed prior to experimentation on a medium that encourages contaminant growth. Accepted: 12 July 1995     

Influence of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> mycelia culture conditions on antimicrobial metabolite production

Enokipodins A, B, C, and D are α-cuparene-type sesquiterpenoids antimicrobial metabolites produced in the stationary stage of Flammulina velutipes mycelia development in malt extract broth. This study assessed the influence of nutritional and environmental factors on F. velutipes mycelia culture for the production of these metabolites. The mycelia growth and antimicrobial activity were assessed by determining dry matter and the diffusion in agar method, respectively. The best F. velutipes mycelia growth was observed in dextrose potato broth, and greater antimicrobial metabolite production occurred in complete Pontecorvo’s culture medium. Environmental modifications, such as a rise in temperature from 25° to 37°C on the 15th day of F. velutipes mycelia culture in malt extract and peptone broth, also optimized antimicrobial metabolite production. The metabolites produced in these treatments were correlated with the enokipodins A and B in thin-layer chromatography (TLC) and the antifungal activity test by TLC bioautography. This study showed that there was no correlation between biomass production and antimicrobial metabolite production, but there may be a correlation between culture medium composition and enokipodins biosynthesis.     

Studies on the survival of corn stalk rot bacteria

Summary Erwinia carotovora andPseudomonas lapsa which cause stalk rot of corn survived in soil and host tissues under different conditions.E. carotovora survived for 150 days and 90 days in sterile and unsterile soils, respectively, whileP. lapsa survived for 135 days and 75 days under similar conditions when they were added as cell suspensions.E. carotovora survived in diseased tissues for 22, 8, and 7 months at 0–5°C (refrigerator), 22–28°C (room), and 20–37°C (field) temperatures whileP. lapsa survived for 26, 10, and 9 months, respectively, under similar conditions. Seeds of Ganga 3 were found to carryP. lapsa for 14 months, when they were stored at room (22–28°C) temperature.     

Ein Beitrag zur axenischen Kultur von zwei insektenbewohnenden Trichomyceten

Zusammenfassung Die TrichomycetenSmittium inopinatum undS. culicis lassen sich erfolgreich auf bei 80°C koagulierten Hühnereidotter kultivieren. Wenn die bei Zimmertemperatur 7 Tage gewachsenen Kulturen anschliessend bei 4–6°C aufgehoben werden, genügt es, Weiterimpfungen nur alle 4 bis 8 Wochen vorzunchmen.S. culicis l?sst sich unter diesen Bedingungen auch auf einem Malzextrakt-Peton-Agar halten.

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Summary Hen's egg yolk coagulated at 80°C proved to be a very suitable culture medium for the TrichomycetesSmittium inopinatum andS. culicis. After growing these cultures for 7 days at room temperature they are subsequently stored at 4–6°C. By this method its is sufficient to make subcultures only at intervals of 4–8 weeks. Under such conditionsS. culicis can be cultivated also on a malt extract peptone agar. — It is possible to storeS. inopinatum for several months at 4° C in soil cultures or in an aqueous solution of 0.675% NaCl.

     

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth from Spores of Nonproteolytic Clostridium botulinum in Heat-Treated Vegetable Juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80°C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80°C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.     

Studies on mould growth and biomass production using waste banana peel

Hyphomycetous (Aspergillus fumigatus) and Phycomycetous (Mucor hiemalis) moulds were cultivated in vitro at room temperature (28 + 20 degrees C) to examined their growth and biomass production on waste banana peel agar (BPA) and broth (BPB) using commercial malt extract agar (MEA) and broth (MEB) as control. The moulds grew comparatively well on banana peel substrates. No significant difference (p > 0.05) in radial growth rates was observed between moulds cultivated on PBA and MEA, although growth rates on MEA were slightly better. Slight variations in sizes of asexual spores and reproductive hyphae were also observed between moulds grown on MEA and BPA. Smaller conidia and sporangiospores, and shorter aerial hyphae (conidiophores and sporangiophores) were noticed in moulds grown on BPA than on MEA. The biomass weight of the test moulds obtained after one month of incubation with BPB were only about 1.8 mg and 1.4 mg less than values recorded for A. fumigatus and M. hiemalis respectively, grown on MEB. The impressive performance of the moulds on banana peel substrate may be attributed to the rich nutrient (particularly the crude protein 7.8% and crude fat 11.6% contents) composition of banana peels. The value of this agricultural waste can therefore be increased by its use not only in the manufacture of mycological medium but also in the production of valuable microfungal biomass which is rich in protein and fatty acids.     

Potential for biological control of Heterodera schachtii by Pochonia chlamydosporia var. chlamydosporia on sugar beet

The biological control potential of an isolate of Pochonia chlamydosporia var. chlamydosporia against Heterodera schachtii was examined by assessing the percentage of females and cysts that became infected on water agar, the effect of culture filtrate on juvenile mobility, and the effects of the fungus on the final population of the nematode on sugar beet under greenhouse conditions. After 3 weeks at 20°C, 74 and 95% of the eggs within cysts and females, respectively, were colonised by the fungus on water agar. The full concentration of the fungal filtrate from cultures in malt extract broth killed only 12% of the juveniles after 24 h at 25°C. In the greenhouse experiment, adding 16,000 chlamydospores of the fungus per gram of soil as either colonised barley grains or spores reduced the final number of females on roots of sugar beet by 50 and 66%, respectively, after 3 months. The reproduction factor was reduced to ×2 in spore-treated soil compared with ×5 in the untreated control, and 18% of the eggs in spore-treated soil were colonised by fungal mycelium. Generally, P. chlamydosporia var. chlamydosporia was more efficient at reducing the nematode population when applied as spores without any substrate than when used as colonised barley grains.     

Heat Activation and Heat-induced Dormancy of Bacillus stearothermophilus Spores

Heat-induced dormancy was observed when spores of two strains of Bacillus stearothermophilus were heated in distilled water at 80, 90, and 100 C. At temperatures above 100 C, true activation occurred; however, maximal activation was not achieved until temperatures of 110 to 115 C were employed. A heat treatment of 115 C for 3 min was required to induce maximal activation in one suspension of strain 1518 spores, whereas a heat treatment of 110 C for 7 to 10 min was adequate for the other suspension of strain 1518 spores. Spores from both strain M suspensions required heat treatments of 110 C for 9 to 15 min for maximal activation. The degree to which the spores could be activated was strain dependent and variable among spore suspensions of the same strain.     

Effects of some chemical factors on flagellation and swarming ofVibrio alginolyticus

Vibrio alginolyticus strains recently isolated from Dutch coastal seawater changed flagellar organization when cultivated in the presence of certain chemical agents. On agar media with more than 4.0% (w/v) NaCl the number of lateral flagella per cell decreased with increasing salt concentration. Both on agar media and in broth cultures with 6.0–9.0% (w/v) NaCl, cells with polar tufts of 2–4 sheathed or unsheathed flagella were frequently found. Cells grown on agar media with 7.3–9.8% (w/v) Na₂SO₄ had drastically reduced numbers of lateral flagella, but lacked polar tufts. EDTA suppressed growth, but did not affect flagellar arrangement. In the presence of 0.1–0.3% boric acid or 0.05–0.1% aluminium hydroxide, cells in liquid media tended to produce lateral, in addition to the polar flagella normally observed in broth cultures. Of a number of surface-active agents tested, Tween 80 and Na-taurocholate, even in high concentrations, did not affect flagellation. Bile salts (0.1%) and Na-deoxycholate (0.05%) strongly reduced the number of both polar and lateral flagella. In agar cultures, Na-lauryl sulphate (0.01–0.1%) inhibited the formation of lateral, but increased the incidence of polar flagella. Teepol (0.05–0.2%) had a similar effect and also it had a deteriorating effect on the sheaths of the polar flagella. Concomitant with the reduction in the number of lateral flagella, induced by these agents, swarming on agar media was inhibited.     

Feasibility of using real-time optical methods for detecting the presence of viable bacteria aerosols at low concentrations in clean room environments

An experimental investigation was carried out to determine the agreement between two methods of viable bacteria aerosol detection. Various amounts of Bacillus globigii (BG) spores were aerosolized in 1-s bursts into a HEPA-filtered air stream and sampled simultaneously with a fluorescence aerosol particle sensor (FLAPS) and a slit to agar biological air sampler. The slit sampler incorporated 150-mm malt extract culture plates, which were incubated at 37°C for at least 12 h before culturable BG particles were counted in terms of colony-forming units (CFU). A relationship between CFU and optically detected viable bacteria particles was determined as culturable particle concentrations decreased. Through further analytical procedures, the FLAPS showed a limit of detection (LOD) of 4.2 bacterial particle/2.5 l of sampled air or 1.7 × 10³ m⁻³. This real-time bacteria aerosol monitor could be used to detect burst contamination events during a surgical procedure. The technology may be used for developing a dose–response relationship between bacterial particle exposure and infection, a tool potentially helpful in determining patient risk.     

Resistance and recovery studies on ultraviolet-irradiated spores of Bacillus pumilus.

A spore suspension model and a procedure for recovering ultraviolet (UV)-irradiated spores of Bacillus pumilus were investigated. A most-probable-number tube dilution method using double-strength Trypticase soy broth was found to be superior to the agar plate method for recovering optimal numbers of spores irradiated with sublethal doses of UV energy. Aqueous suspensions of B. pumilus survived UV doses up to 108,000 ergs/mm2 as determined by a most-probable-number recovery and estimation procedure. Resistance and stability data were consistent and reproducible, indicating the dependability of this method for recovering UV-damaged spores. The procedures used to collect information concerning resistance characteristics for two strains of B. pumilus are discussed.     

Growth from spores of nonproteolytic Clostridium botulinum in heat-treated vegetable juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80 degrees C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80 degrees C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.     

Penicillium discolor, a new species from cheese, nuts and vegetables

The new species Penicillium discolor, frequently isolated from nuts, vegetables and cheese is described. It is characterised by rough, dark green conidia, synnemateous growth on malt agar and the production of the secondary metabolites chaetoglobosins A, B and C, palitantin, cyclopenin, cyclopenol, cyclopeptin, dehydrocyclopeptin, viridicatin and viridicatol. It also produces the mouldy smelling compounds geosmin and 2-methyl-isoborneol, and a series of specific orange to red pigments on yeast extract sucrose agar, hence the epithet discolor. P. discolor resembles P. echinulatum morphologically but on basis of the secondary metabolites is also related to P. expansum, P. solitum and P. crustosum.     

Cryopreservation at −80°C of Agaricus blazei on rice grains

The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After 1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among the strains of this fungus do not interfere in the cryopreservation process.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.