Revival of saprotrophic and mycorrhizal basidiomycete cultures after 20 years in cold storage in sterile water

Vegetatively colonized agar cores of 69 basidiomycete fungus isolates (48 species in 30 genera and 17 families) were stored at 5 degrees C in tubes of sterile distilled water without manipulation for 20 years. These were represented by 34 isolates of saprotrophic fungi (29 species in 19 genera) and 35 isolates of mycorrhizal fungi (19 species in 11 genera). Viability was evaluated based on revived growth on agar media at room temperature. Fifty-seven of the 69 isolates (82.6%) grew vigorously when revived after storage for 20 years; of the 34 saprotrophic fungus isolates, 30 revived (88.2%); of the 35 mycorrhizal fungus isolates, 27 revived (77.1%). Thirteen isolates of Laccaria were all viable after 20 years, indicating cold storage in sterile water to be a good method for maintaining this important genus of mycorrhizal fungi. In general, however, mycorrhizal fungus species demonstrated lower viability than saprotrophic fungi.     

Evaluation of the Viability of Pathogenic Filamentous Fungi after Prolonged Storage in Sterile Water and Review of Recent Published Studies on Storage Methods

We have evaluated the survival and potential morphological alterations of 45 species of pathogenic filamentous fungi that had been stored in sterile water following Castellani’s method in the National Collection of Pathogenic Fungi (NCPF). Storage duration varied from 2 months to over 21 years. Ninety percent of stored organisms were shown to be viable. Viability was largely independent of the duration of storage, but did apparently vary to some degree in an organism-specific manner. In addition, certain fungi were shown to have undergone morphological alterations during storage, and exhibited significant degrees of pleomorphism upon revival. This was especially marked for several isolates of dermatophytes, where storage resulted in loss of recognisable colonial features, and overproduction of sterile mycelium with aberrant or no conidia. These findings suggest that while Castellani’s method remains an easy and inexpensive method for long-term preservation of most fungi, water storage should be supplemented by a second storage method to increase the chances of retaining both viability and morphological stability over long periods.     

Screening for Indian isolates of egg-parasitic fungi for use in biological control of fascioliasis and amphistomiasis in ruminant livestock

Wild isolates of the egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium, obtained from the organic environment of Durg, Chhattisgarh, India, were subjected to screening for in vitro growth using different media types, range of incubation temperature and pH, and their predatory activity to the eggs of Fasciola gigantica and Gigantocotyle explanatum. Maximum growth of P. lilacinus was obtained in corn-meal agar compared to any other media types. The preferred medium for growth of V. chlamydosporium was corn-meal agar, followed by potato-dextrose agar. After initial growth for 16 h of incubation, no growth was observed in water agar for both the fungi. Six different temperatures--4 degrees C, 10 degrees C, 18 degrees C, 26 degrees C, 34 degrees C and 40 degrees C--were used to observe growth profiles of the fungi in corn-meal agar medium. While no and very little growth of P. lilacinus and V. chlamydosporium was observed at 4 degrees C and 10 degrees C, respectively, growth profiles of both the fungi were optimal at 26-40 degrees C. A range of pH (pH 4-8) supported growth of both P. lilacinus and V. chlamydosporium. Full-grown plates of the fungi baited with viable eggs of F. gigantica and G. explanatum revealed that V. chlamydosporium was more vigorous in its egg-parasitic ability compared to P. lilacinus. Distortion of the eggs started on day 2-3 of egg baiting in culture plates of V. chlamydosporium, with complete distortion by day 7. On the contrary, P. lilacinus exhibited very limited egg-parasitic ability and some of the baited eggs even showed development of miracidia.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

Long-term maintenance of fungal cultures on perlite in cryovials — An alternative for agar slants

Cultures of 33 basidiomycete strains out of 35 tested were viable with unchanged characteristics after four years of maintenance on perlite in cryovials. These cultures can be a good substitute for agar cultures in long-term maintenance of fungi. For comparison, the storage under oil was evaluated but it turned out to be unsuitable for the majority of our cultures.     

Characterization of Sphingomonas isolates from Finnish and Swedish drinking water distribution systems

Sphingomonas species were commonly isolated from biofilms in drinking water distribution systems in Finland (three water meters) and Sweden (five water taps in different buildings). The Sphingomonas isolates (n = 38) were characterized by chemotaxonomic, physiological and phylogenetic methods. Fifteen isolates were designated to species Sphingomonas aromaticivorans, seven isolates to S. subterranea, two isolates to S. xenophaga and one isolate to S. stygia. Thirteen isolates represented one or more new species of Sphingomonas. Thirty-three isolates out of 38 grew at 5 degrees C on trypticase soy broth agar (TSBA) and may therefore proliferate in the Nordic drinking water pipeline where the temperature typically ranges from 2 to 12 degrees C. Thirty-three isolates out of 38 grew at 37 degrees C on TSBA and 15 isolates also grew on blood agar at 37 degrees C. Considering the potentially pathogenic features of sphingomonas, their presence in drinking water distribution systems may not be desirable.     

Long-term survival of isolates of various Cladosporium and Fusicladium species under mineral oil

Lima bean agar cultures of several Cladosporium and Fusicladium species were placed under sterile mineral oil and stored in a refrigerator in 1956. Isolates of C. herbarum, C. fulvum, C. cucumerinum, and F. dendriticum were found to be non-viable in 1983 (after 27 years), but an isolate of C. carpophilum was found to be still viable. Earlier, in 1980, several isolates of C. caryigenum (syn. F. effusum and C. effusum) were found to be viable and capable of sporulating on various agar media (after 24 years). In 1983, re-oiled cultures of surviving C. caryigenum isolates were tested for pathogenicity to its host (Carya illinoensis — the pecan tree). One isolate was found to be still pathogenic.     

Experimental studies on the persistence in distilled water of certain conditional pathogenic fungi

The authors followed up the behaviour in distilled water of 8 fungus strains belonging to four species (Candida albicans, C. krusei, Geotrichum candidum and Rhodotorula) kept at various temperatures (i.e. +4 degrees, +18 degrees and 25 degrees C). The average and the maximum survival periods were found to be as follows: C. albicans--476/1138 days, C. krusei--411/1138 days, G. candidum--127/219 days, and R. rubra--139/226 days. All strains revealed periodically multiplication bursts of variable intensity depending on the kind of species and the temperature at which the cultures were maintained. These findings plead for the existence of cryptic periods of growth in fungi, too. Although, during the experiments, the variants occurred as concerns the morphological and cultural characteristics, sugar fermentation, sugar and nitrate assimilation, germ tube formation did not undergo any modifications. The pathogenic characteristics of C. albicans strains for rabbit were retained even after 1096 days maintenance in distilled water at 18 degrees C.     

Preservation, mass production and storage ofStagonospora convolvuli, a bioherbicidecandidate for field bindweed (Convolvulusarvensis)

Different solid substrates were investigated as spore production methods for Stagonospora convolvulistrain LA39, a potential bioherbicide for field bindweed (Convolvulusarvensis L.). Up to 4 × 10⁸ spores/g of substratewere yielded on cous-cous (cracked hard wheat). Thespores were as pathogenic as those grown on artificial medium (V-8-juice agar). The air-drying on kaolin and storage at 3 °C kept spores viable and pathogenic for 180 days. Spore germination exceeded70% for the first 140 days and then declined to 50%after 175 days. Less than 5% of spores were still viable after 17 months. The preservation of stock cultures in 10% glycerine at −80 ° C and in liquid nitrogen did not affect viability orpathogenicity of the spores. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phialophora parasitica, an emerging pathogen

Monoconidial cultures derived from 12 clinical and environmental isolates of Phialophora parasitica were compared with respect to morphologic and physiologic characteristics and response to antifungal agents. No yeast cells were seen in 1- and 3-week-old slide culture preparations. Also, not all of the distinguishing characteristics of this species were displayed by all isolates on all media examined. Although the isolates grew on Sabouraud agar with chloramphenicol and cycloheximide, some inhibition was observed. All cultures were strongly urease-positive and hydrolyzed casein and starch; most decomposed tyrosine but not gelatin. All but one environmental isolate grew well at both 23 and 37 degrees C, but none grew at 40 degrees C. In the sensitivity testing the isolates did not vary much in their response to each drug, although some anomalies were observed. Amphotericin B and miconazole had minimum inhibitory concentrations in the low sensitivity range (2.0-8.0 and 2.5-10 micrograms m-1 respectively), for most isolates, and most isolates were resistant to both 5-fluorocytosine and ketoconazole. Limited observations were made on three other Phialophora species which might be confused with P. parasitica.     

Survival of Campylobacter jejuni strains of different origin in drinking water

AIMS: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. METHODS AND RESULTS: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. CONCLUSIONS: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in culturability depending on culture conditions and on strain origin.     

The effects of temperature on growth of four high Arctic soil fungi in a three-phase system

The effect of temperature on the growth of Chrysosporium pannorum, Cylindrocarpon sp., Penicillium janthinellum, and Phoma herbarum, isolated from tundra soils, was studied. The growth in two systems, glucose-mineral agar plates and sand, moistened with glucose-mineral broth, was compared. All isolates showed an exponential increase in mass (measured as protein increase) in sand and a linear rate of extension on agar. Radial increase on agar was shown not to be a good index of growth in sand. Trends in growth rates in the sand cultures indicated that all four fungi can grow at low temperatures. The growth rate for Penicillium janthinellum at 15 degrees C was higher than at 20 degrees C, and Cylindrocarpon sp. and Phoma herbarum had higher growth rates at 2.5 degrees C than at 5 degrees C. These data suggest that there may be some adaptation by these fungi to growth in Arctic regions.     

Preservation of in vitro cultures of the mite pathogenic fungus Neozygites tanajoae

Neozygites tanajoae has recently been described as a new fungal pathogen distinct from Neozygites floridana. This pathogen is currently being used as a classical biological control agent against the cassava green mite, Mononychellus tanajoa (Bondar), in Africa. Neozygites tanajoae is a particularly fastidious species, and in vitro cultures of isolates from Brazil and Africa have only recently been established. In this study, the efficacy of several cryoprotectants at different exposure times, cooling rates, and warming rates for protecting hyphal bodies of N. tanajoae during cryopreservation was investigated. A protocol for preservation of cultures of N. tanajoae at ultra-low temperatures of -80 degrees C or -196 degrees C, using 1% trehalose + 2% dimethyl sulfoxide as cryoprotective agents, is described in detail. In this study, we demonstrate that N. tanajoae differs remarkably from N. floridana (isolates ARSEF 662 and ARSEF 5376) in the ability to withstand the stress of cold temperature (4 degrees C) and cryopreservation. In vitro cultures of the 2 N. floridana isolates remained viable at 4 degrees C for up to 47 d; however, cultures of N. tanajoae did not survive this temperature for 4 d. Cryopreservation methods successful for N. tanajoae isolates are not suitable for N. floridana and are unusual in comparison to those for many fungi.     

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.     

Survival of Filamentous Fungi in Hypersaline Dead Sea Water

A variety of filamentous fungi have recently been isolated from the Dead Sea (340 g/L total dissolved salts). To assess the extent to which such fungi can survive for prolonged periods in Dead Sea water, we examined the survival of both spores and mycelia in undiluted Dead Sea water and in Dead Sea water diluted to different degrees with distilled water. Mycelia of Aspergillus versicolor and Chaetomium globosum strains isolated from the Dead Sea remained viable for up to 8 weeks in undiluted Dead Sea water. Four Dead Sea isolates (A. versicolor, Eurotium herbariorum, Gymnascella marismortui, and C. globosum) retained their viability in Dead Sea water diluted to 80% during the 12 weeks of the experiment. Mycelia of all species survived for the full term of the experiment in Dead Sea water diluted to 50% and 10% of its original salinity. Comparison of the survival of Dead Sea species and closely related isolates obtained from other locations showed prolonged viability of the strains obtained from the Dead Sea. Spores of isolates obtained from the terrestrial shore of the Dead Sea generally proved less tolerant to suspension in undiluted Dead Sea water than spores of species isolated from the water column. Spores of the species isolated from the control sites had lost their viability in undiluted Dead Sea water within 12 weeks. However, with the exception of Emericella spores, which showed poor survival, a substantial fraction of the spores of Dead Sea fungal isolates remained viable for that period. The difference in survival rate between spores and mycelia of isolates of the same species points to the existence of adapted halotolerant and/or halophilic fungi in the Dead Sea.     

Storage of Stock Cultures of Filamentous Fungi, Yeasts, and Some Aerobic Actinomycetes in Sterile Distilled Water

Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable.     

Isolation, Growth Characteristics, and Long-Term Storage of Fungi Cultivated by Attine Ants

Seven pure-culture strains of fungi cultivated by attine ants (ant-garden fungi) were isolated from locally maintained leaf-cutting ant colonies. An ant-garden fungus strain obtained from an Atta cephalotes colony, when offered to ants of the colony from which the fungus was isolated, was accepted as their own. Young fungus cultures were harvested and incorporated into the fungus garden, and cultures of intermediate age were used to begin a new fungus garden; old cultures were simply harvested. To facilitate further research on this fungus, growth characteristics of the different isolates were studied under a variety of conditions. They grew better at 24°C than at 30°C, and growth did not occur at an incubation temperature of 37°C. In a broth culture medium, growth was enhanced by aeration of the culture and by addition of yeast extract, olive oil, sesame oil, peanut oil, soybean oil, corn oil, sunflower oil, cottonseed oil, walnut oil, safflower oil, or mineral oil. Glycerol did not noticeably affect growth, but Tween 80 inhibited growth. These fungi were extremely sensitive to cycloheximide, growth being totally inhibited at cycloheximide concentrations ranging from 0.4 to 4.0 μg/ml. To date, the ant-garden fungus isolates have remained viable in long-term mineral oil-overlay storage cultures for up to 4 years.     

Identification of Vibrio spp. (other than V. vulnificus) recovered on CPC agar from marine natural samples.

Two hundred and eighty four presumptive but not confirmed Vibrio vulnificus isolates grown on cellobiose-polymixin B-colistin agar (CPC) at 40 degrees C, recovered from sea water samples from Valencia, Spain, during a microbiological survey for V. vulnificus, were phenotypically identified. Most of the isolates (91%) corresponded to Vibrio species. V. harveyi (24%) and V. splendidus(19%) were the most abundant species identified, followed by V. navarrensis (13%), V. alginolyticus (8%) and V. parahaemolyticus (5%). The ability to grow on CPC agar and ferment cellobiose of several V. vulnificus strains from different origins and serovars, including reference strains, was tested. Most serovar E isolates and 25% of non-serovar E isolates could not grow on CPC agar.     

Effects of indole-3-acetic acid on Botrytis cinerea isolates obtained from potted plants

We study the growth of different isolates of Botrytis cinerea collected from potted plants which were affected by Botrytis blight in southern Spain during recent years. These isolates, which show widely phenotypic differences when grown in vitro, are differentially affected by growth temperature, gibberellic acid applications and paclobutrazol, an efficient plant growth retardant and fungicide at the same time. In this work, we have evaluated the effect of the auxin indole-3-acetic acid (IAA) dose (0, 1, 10, and 100 mg/plate) on the growth of the collection of B. cinerea isolates obtained from the following potted plants: Cyclamen persicum, Hydrangea macrophylla, Lantona camara, and Lonicera japonica. B. cinerea produces indolacetic acid, but so far the precise biosynthetic pathway and some effects on this fungal species are still unclear, although recent studies have revealed an antifungal activity of IAA on several fungi, including B. cinerea isolated from harvested fruits. Mycelial growth curves and growth rates assessed from difference in colony areas during the both linear and deceleration phase, conidiation (measured as time of appearance), conidia length (microm), and sclerotia production (number/plate) were evaluated in the isolates, which were grown at 26 degrees C on Petri dishes containing potato dextrose agar for up to 35 days. Mycelial growth curves fitted a typical kinetic equation of fungi grown on solid media. B. cinerea isolates showed a high degree of variability in their growth kinetics, depending on the isolate and auxin dose. This plant growth substance delayed mycelial growth during the linear phase in an isolate-dependent manner, thus isolates from C. persicum, H. macrophylla and L. camara were more affected by IAA than L. japonica. On the other hand, 100 mg of IAA was the critical dose to significantly reduce the growth rate in all isolates and to promote brown-striped hyphae development, especially in isolate from C. persicum. 10 and 100 mg IAA delayed conidiation in isolates from H. macrophylla but scarcely effects were found in the conidia length. The sclerotia production process was blocked at IAA doses of 100 mg in isolates from L. camara and L. japonica, and was reduced in isolate from H. macrophylla. However, dose of 100 mg IAA had no effect on sclerotia production in isolate from C. persicum. It was concluded that the effect of IAA on B. cinerea growth depends on the isolate, thus isolates from H. macrophylla and L. camara were the most affected by IAA. B. cinerea reduced its development under IAA applications, depending on the isolate and dose. These results confirm those recently published on the inhibitory effect of IAA on Botrytris species growth.     

Relative sensitivities of environmental legionellae to selective isolation procedures

A survey of water samples to determine the efficacy of standard procedures for the isolation of environmental legionellae was conducted. Marked variations in intraspecies resistance to selective agents and treatments were observed, and in experiments with one of the isolates, the response was modified by culture conditions. Five selective procedures incorporating acid (pH 2.2) and heat (50 degrees C, 30 min) treatments, with and without plating on buffered charcoal-yeast extract agar supplemented with vancomycin (5 micrograms/ml), polymyxin B (60 U/ml), and cycloheximide (80 micrograms/ml), caused 5 to 99% decreases in viable counts of pure cultures in water suspensions. The differences in the responses of the cultures to the five treatments were statistically significant. Cells in retained samples of naturally contaminated water from which the original cultures had been isolated were significantly less sensitive than artificially grown isolates. The sensitivities of the laboratory-grown cells to the treatments were affected by the length of incubation on buffered charcoal-yeast extract agar. Whereas acid resistance increased after 24 h of incubation, resistance to the antibiotic mixture decreased.