Production of fumonisins B<Subscript>2</Subscript> and B<Subscript>4</Subscript> in <Emphasis Type="Italic">Tolypocladium</Emphasis> species

Tolypocladium inflatum is known primarily for its production of the cyclosporines that are used as an immunosuppressive drug. However, we report here the production of the carcinogenic fumonisins B₂ and B₄ by this biotechnologically relevant fungal genus. These mycotoxins were detected in 11 strains tested from three species: Tolypocladium inflatum, T. cylindrosporum, and T. geodes. Production of fumonisins by Fusarium spp. and Aspergillus niger is highly medium- and temperature-dependent, so the effect of these parameters on fumonisin production by three T. inflatum strains was studied. Maximum production was achieved on media with high sugar content incubated at 25–30°C. Since these results demonstrate that fumonisin production could be widespread within the genus Tolypocladium, the potential contamination of commercial cyclosporine preparations with fumonisins needs to be investigated.     

Conidial water affinity is an important characteristic for thermotolerance in entomopathogenic fungi

The interspecific thermotolerance of several species of entomopathogenic fungi was evaluated based on the conidial water affinity. The species were divided between hydrophilic and hydrophobic conidia. The species with hydrophobic conidia were Beauveria bassiana (ARSEF 252), Metarhizium brunneum (ARSEF 1187), Metarhizium robertsii (ARSEF 2575), Isaria fumosorosea (ARSEF 3889) and Metarhizium anisopliae s.l. (ARSEF 5749). The species with hydrophilic conidia were Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), Simplicillium lanosoniveum (ARSEF 6430), Lecanicillium aphanocladii (ARSEF 6433), S. lanosoniveum (ARSEF 6651), Aschersonia placenta (ARSEF 7637) and Aschersonia aleyrodis (ARSEF 10276). The conidial surface tension of each isolate was also studied. Conidial suspensions were exposed to 38, 41 or 45 °C. After exposure, the suspensions were inoculated on media and conidial germination was evaluated. Considerable differences in thermotolerance were found among the 12 entomopathogenic fungal species. Species with hydrophobic conidia were generally more thermotolerant than species with hydrophilic conidia. All isolates with hydrophobic conidia showed higher conidial surface tension than the isolates with hydrophilic conidia.     

Tolypocladium cylindrosporum gams (deuteromycetes,moniliaceae) as a biological control agent against the window midge Sylvicola cinctus Fabr. (diptera: Anisopodidae)

Larvae of Sylvicola cinctus Fabr. were found killed by an entomopathogenic fungus Tolypocladium cylindrosporum Gams, in Vetlanda, Sweden. This is the first report of this fungus as an insect pathogen from Sweden. The fungus was cultured and sprayed on aspen log piles in an attempt to reduce population levels. In September 1992, when maximum abundance is expected, the area treated with T. cylindrosporum had significantly fewer midges than an untreated area. Possibilities for biological control are discussed.     

Cyclosporin-A production by Tolypocladium inflatum using solid state fermentation

Tolypocladium inflatum strains are known to produce Cyclosporin-A under submerged culture conditions. In the present study solid state fermentation was used to produce Cyclosporin-A. Tolypocladium inflatum strains when grown on moist wheat bran produced 310–459 mg of Cyclosporin-A kg of bran⁻¹. Tolypocladium inflatum ATCC 34921 which produced 459 mg of Cyclosporin-A kg of bran⁻¹ was improved to produce 1031±27 mg of Cyclosporin-A kg⁻¹ of bran, by subjecting the spores to different mutagenic treatments. The mutated strain, designated Tolypocladium inflatum DRCC 106, produced 4843 mg kg⁻¹ of bran under optimum fermentation conditions in 10 days when grown on wheat bran medium containing millet flour 20%, jowar flour 10%, zinc sulphate 0·15%, ferric chloride 0·25% and cobaltous chloride 0·05%. An inoculum of 60% initial moisture content 70%, initial bran pH 2·0 and incubation temperature 25°C were found to be optimal. Cyclosporin-A thus obtained was purified by solvent extraction, followed by column chromatography. The isolated product complies with the United States Pharmacopoeia specifications.     

Tolypocladin — a new metal-chelating 2-aza-anthraquinone fromTolypocladium inflatum

Summary A new chelator of di- and trivalent cations (tolypocladin) was isolated from the mycelium ofTolypocladium inflatum DSM 915. The structure has been determined by NMR methods as 3-methyl-5,6(7),8-trihydroxy-2-aza-anthraquinone. The ultraviolet, visible and fluorescence spectral properties of some metal complexes (in methanol) are described. The compound forms water-soluble fluorescent aluminium complexes. Its production is dependent on zinc ions in the medium. It serves as an endogenous hydrogen acceptor under oxygen limitation in the producing strain,T. inflatum.     

Dominating species of entomophilous ascomycetes anamorphs in West Siberia,Primorsky krai,and Kyrgyzstan

The paper studies mycobiota of the dead insects in West Siberia, Primorsky krai, and Kyrgyzstan. Ascomycetes anamorphs of 13 genera are revealed. In all regions Beauveria bassiana (Bals.-Criv.) Vuill. dominated comprising on average 68% of the total number of isolates. The fungus hosts list the insects of 7 orders and 32 families with Coleoptera, Lepidoptera, and Hemiptera dominating. The rarely found entomopathogens include Tolypocladium inflatum Gams (primarily on Lepidoptera), Metarhizium anisopliae (Metschn.) Sorokin (on Coleoptera). The mortality rate of the insects due to micromycetes is observed mainly on enzootic level. The study of the pathogenic properties of the dominating species (B. bassiana) show the absence of specificity of its environmental isolates for a number of representatives of Orthoptera, Lepidoptera, Coleoptera, and Diptera.     

Cold activity and tolerance of the entomopathogenic fungus Tolypocladium spp. to UV-B irradiation and heat

Studies on the stress resistance of insect-pathogenic fungi are very important to better understand the survival of these organisms in the environment. In this study, we examined the cold activity (8 ± 1 °C for 7 days), UV-B tolerance (Quaite-weighted UV-B irradiance at 847.90 mW m⁻² for 1, 2, 3, and 4 h), and wet-heat tolerance (45 °C for 1, 2, 3, and 4 h) of two isolates of Tolypocladiumcylindrosporum (ARSEF 3392 and 5558), one isolate of Tolypocladium geodes (ARSEF 3275), and two isolates of Tolypocladium inflatum (ARSEF 4772 and 4877) based on their germination, compared with Metarhizium robertsii (ARSEF 2575). After 3 h of UV-B exposure, T. cylindrosporum germinated at a greater rate than the other Tolypocladium species and had similar viability to that of the M. robertsii. Most Tolypocladium isolates, however, were less UV-B tolerant than M. robertsii. The T.cylindrosporum isolates were also the most thermotolerant, with similar tolerance to the M. robertsii. The isolates of T. inflatum and T. geodes, which had similar heat tolerance, were the least heat tolerant compared with the isolates of T. cylindrosporum and M. robertsii. After 4 h of heat exposure, the germination of T. inflatum and T. geodes isolates was not significantly different. For cold activity, both T.cylindrosporum isolates germinated to ca. 100% in only 3 days. Approximately 50% of the two T. inflatum isolates germinated, and less than 5% of T. geodes germinated after 3 days. All fungal isolates, however, completely germinated by the seventh day, except M.robertsii. The isolates of T. cylindrosporum, therefore, were the most heat and UV-B tolerant, and had the highest cold activity compared to the other species. The tolerance of M. robertsii to UV-B radiation and heat was similar to that of T.cylindrosporum.     

Growth and cyclosporin a production by an indigenously isolated strain ofTolypocladium inflatum

A fungal culture isolated from a local soil sample which showed antifungal activity and produced cyclosporin A, was identified asTolypocladium inflatum. The culture grew best in a medium containing 1% maltose (pH 5–6) when inoculated with a one-day-old inoculum at 2% (V/V) concentration. Under batch fermentation conditions, growth and cyclosporin A production were better in complex media (24.6 g biomass and 205 mg cyclosporin A per liter) in comparison with synthetic media (6.84 g biomass and 35 mg cyclosporin A per liter). While addition of peptone increased culture growth (high biomass yield), supplementation with casein acid hydrolyzate favored cyclosporin A production.     

Entomopathogenic species of the hyphomycete genus Tolypocladium

Two entomopathogenic species of the hyphomycete genus Tolypocladium are described in detail. Tolypocladium extinguens sp. nov. was found on larvae of Arachnocampa luminosa (Diptera; Mycetophilidae) in caves in New Zealand. Tolypocladium cylindrosporum W. Gams is shown to be pathogenic to Aedes sierrensis and A. australis and is reported from Plecia nearctica (Diptera; Bibionidae). The occurrence of entomopathogenic species in this normally soil-borne genus is discussed.     

Pigmented variants of Tolypocladium inflatum in relation to cyclosporin A production

Summary We have isolated four distinct colony types of the fungus Tolypocladium inflatum, the producer of the immunosuppresive agent cyclosporin A: morphologically normal white, red, and orange colonies and morphologically diverse tiny brown colonies. In liquid cultures, white normal and brown colonies developed into yellow broths. The broth of the brown colony had a low final pH and low cyclosporin production, whereas orange and red colonies had dark brown and even black broths with higher final pH and high cyclosporin production. The specific production of cyclosporin A by the red colony was three times that of the white normal colonies. Offprint requests to: S. N. Agathos     

Comparative studies of eleven isolates of the fungal entomopathogen Tolypocladium cylindrosporum and two isolates of Tolypocladium extinguens

The virulence of 11 isolates of Tolypocladium cylindrosporum and 2 isolates of Tolypocladium extinguens was evaluated against mosquito larvae. Second-instar Aedes aegypti larvae were more susceptible to infection by blastospores than were second-instar Anopheles stephensi larvae. Only the T. extinguens isolates were not infectious for A. aegypti or A. stephensi. Nine of the remaining strains killed 100% of larvae at the highest dose tested (10⁶ blastospores/ml). In vitro sporulation and conidiospore size were also studied. Significant differences were observed for both characteristics. Characterization of strains according to in vitro sporulation and virulence made it possible to select the most promising strains for future laboratory and field tests against mosquitoes.     

Tick pathogenicity,thermal tolerance and virus infection in Tolypocladium cylindrosporum

Tolypocladium cylindrosporum is a fungus which has been isolated from soil, from asymptomatic plants as an endophyte and has been shown to be pathogenic to several species of arthropods. The objective of the present work was to study a collection of T. cylindrosporum strains to evaluate the characteristics of this fungus as a bioacaricide. The pathogenicity of five different strains of T. cylindrosporum was tested against two tick species, Ornithodoros erraticus and Ornithodoros moubata. Both tick species were susceptible to all the fungal strains. Mortality was greater for O. erraticus and differed among the five developmental stages of the ticks tested and among the fungal treatments. Mean mortality rates were close to 60% for O. erraticus, similar to those reported for other entomopathogenic fungi used for this purpose. The responses of 11 different strains of the fungus to 22°C and 30°C were also studied. Significant differences in temperature tolerance occurred among the strains and growth inhibition was observed at 30°C. Several mycoviruses were found infecting 5 of the 11 strains. However, no clear relationship was found between the presence of viruses and fungal growth or pathogenicity.     

Physiological and genetic factors for process development of cyclosporine fermentations

Summary The new immunosuppressive agent cyclosporine (Cyclosporin A, Cy) is the most prominent member of a group of cyclic peptide fungal metabolites (cyclosporins) produced byTolypocladium inflatum in submerged fermentations. In the present study, kinetics and physiology of mycelial growth and Cy production byT. inflatum were examined. A new semi-synthetic medium was formulated, consisting of a single carbon/energy source, Bacto-peptone, potassium phosphate and potassium chloride. A wide variety of carbon sources supported growth and Cy production. 3% (w/v) sorbose gave the highest final Cy titer (105.5 mg/l), based on 10-day fermentations. The best specific Cy production was observed with 2% sorbose (14.3 mg Cy/g biomass) followed by 5%myo-inositol (13.4 mg Cy/g biomass). A feeding strategy consisting of sequential addition of two carbon sources such as sorbose and maltose was developed in order to reach higher volumetric production. Genetic studies were also conducted, focussing on the development of mutants for increased Cy production and for the synthesis of novel cyclosporins. In the course of these studies, viable protoplasts ofT. inflatum have been isolated and regenerated.     

rRNA sequence comparison of Beauveria bassiana, Tolypocladium cylindrosporum, and Tolypocladium extinguens

Five strains of Tolypocladium cylindrosporum, one strain of Tolypocladium extinguens, and nine strains of Beauveria bassiana were analyzed using a rapid rRNA sequencing technique. The sequences of two highly variable domains (D1 and D2) located at the 5' end of the 28S-like rRNA molecule were determined. The phylogenetic tree computed from the absolute number of nucleotide differences shows the separation between the genus Beauveria and the genus Tolypocladium and points out that T. cylindrosporum and T. extinguens probably do not belong to the same genus.     

Effect of amino acids on the production of cyclosporin a byTolypocladium inflatum

Summary Cyclosporin A was produced in semi-synthetic and synthetic media byTolypocladium inflatum in suspension culture. L-valine increased the specific production of cyclosporin A by 62% in semi-synthetic media and by 4 times in synthetic media, compared to an unsupplemented control culture. D-valine had no stimulatory effect on the production. L-leucine was also a strong enhancer of drug production in synthetic media. Experiments with different times of addition of L-valine indicated that the amino acid may need to be present in exponential growth phase for optimal production. 4g/l was the optimum concentration of exogenous L-valine. In contrast, exogenous L-methionine and sarcosine tended to diminish the production of cyclosporin A.     

Comparative study reveals unique features of the mycobiota in peat soils samples from Japan and Scotland

We isolated filamentous fungi from soil samples of peat layers in Aomori and Oita Prefectures in Japan and Perth and Kinross district in Scotland by a serial dilution plate technique. The mycobiota in each peat soil showed some common and characteristic features. The abundance of fungal isolates (CFU/g) from peat soil was low: about 1/3 to 1/30 compared with evergreen or coniferous forests or cultivated soil. Trichoderma or Mucorales species were scarcely observed; these fungi occupied only 3% of the total number of colonies. On the other hand, fungi such as Conioscypha and Tolypocladium that are normally isolated rather rarely were encountered at a comparatively high rate. Acremonium guillematii and Tolypocladium cylindrosporum were recorded for the first time in Japan. Sterile fungi occupied 50% of the total number of isolates. The low abundance of fast-growing fungi enabled us to pick slow-growing fungi up easily from the isolation medium. It is interesting that species not previously described in Japan, or scarcely reported, were isolated commonly from both Japanese and Scottish samples. A peat soil sample is therefore an attractive source of untapped microbial resources.     

Tolypocladium cylindrosporum (Deuteromycotina: Moniliales), a fungal pathogen of the mosquito Aedes australis

The New Zealand strain of Tolypocladium cylindrosporum was cultured on Sabouraud dextrose agar medium under varying regimes of growth conditions. The isolate exhibited good tolerances to temperature (4–35 °C), pH (3–10) and salinity (0–7% NaCl). Optimal vegetative growth and sporulation were recorded between a temperature range of 20–30° C, pH of 5–6 and a salinity level of 0–2% NaCl. The North American isolate of the fungus showed similar tolerances, while the European isolate was less tolerant.     

Rheology and Hydrodynamic Properties of Tolypocladium inflatum Fermentation Broth and Its Simulation

A physico-chemical, two phase simulated pseudoplastic fermentation (SPF) broth was investigated in which Solka Floc cellulose fibre was used to simulate the filamentous biomass, and a mixture of 0.1% (w/v) carboxymethyl cellulose (CMC) and 0.15 M aqueous sodium chloride was used to simulate the liquid fraction of the fermentation broth. An investigation of the rheological behaviour and hydrodynamic properties of the SPF broth was carried out, and compared to both a fungal Tolypocladium inflatum fermentation broth and a CMC solution in a 50 L stirred tank bioreactor equipped with conventional Rushton turbines. The experimental data confirmed the ability of the two phase SPF broth to mimic both the T. inflatum broth bulk rheology as well as the mixing and mass transfer behaviour. In contrast, using a homogeneous CMC solution with a similar bulk rheology to simulate the fermentation resulted in a significant underestimation of the mass transfer and mixing times. The presence of the solid phase and its microstructure in the SPF broth appear to play a significant role in gas holdup and bubble size, thus leading to the different behaviours. The SPF broth seems to be a more accurate simulation fluid that can be used to predict the bioreactor mixing and mass transfer performance in filamentous fermentations, in comparison with CMC solutions used in some previous studies.     

Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless

Restless is an endogenous hAT transposon found in the cyclosporin-producing fungus Tolypocladium inflatum. This element is present in about 15 copies in a particular strain (ATCC34921) which was used for successful gene tagging. We have isolated a T. inflatum mutant with a defect in nitrogen metabolism. This mutant carries a copy of the Restless element in a gene encoding a C6 zinc-finger protein. The deduced amino acid sequence of the gene product shows a significant similarity to the NIT4 protein of Neurospora crassa, which is a regulator of nitrogen metabolism. The wild-type T. inflatum gene was shown to complement a nit-4 mutant of N. crassa. From these data, we conclude that the T. inflatum gene also encodes a regulator of nitrogen metabolism, which was named tnir1 (Tolypocladium nitrogen regulator 1). To the best of our knowledge, this is the first fungal gene to be identified by transposon-directed gene tagging. A general method for gene tagging using an endogenous fungal transposon is presented. Received: 20 October 1999 / Accepted: 15 December 1999