Mycelial Fragments Research Articles

Spread of Phytophthora Root and Crown Rot in Saintpaulia, Gerbera and Spathiphyllum Pot Plants in ebb-and-flow-systems

Spread of Phytophthora root and crown rot in three pot plant species was studied on ebb-and-flow benches where the nutrient solution was recirculated. The plant species and their respective pathogens were: Saintpaulia ionantha – P. nicotianae, Gerbera jamesonii – P. cryptogea, and Spathiphyllum wallissii – Phytophthora spp. Ebb-and-flow benches were infested with the pathogen using different methods: 18–25% of the plants on a bench were inoculated or potted in soil infested with the pathogen or the nutrient solution was infested by either zoospores or mycelium fragments. More than 80% of the inoculated Saintpaulia plants and 22% of plants potted in infested soil developed disease but no spread of the disease was observed. Infestation of the nutrient solution did not result in any diseased Saintpaulia plant. More than 70% of the Gerbera plants developed disease as a result of spread of the pathogen irrespective of the infestation method used. No significant spread of the disease was observed with inoculated Spathiphyllum plants nor from plants potted in infested soil. A few Spathiphyllum plants developed disease symptoms after infestation of the nutrient solution with zoospores. In one experiment, nearly all Spathiphyllum plants were diseased after infestation of the nutrient solution with mycelium fragments. The presence of an irrigation mat significantly reduced the spread of the Phytophthora disease in Gerbera and Spathiphyllum. The possibility of an irrigation mat acting as a filter for zoospores is discussed.

Effect of ecological factors on spore germination and the viability of the mycelial fragments of microscopic fungi

Spore germination and the viability of the mycelial fragments of the microscopic fungi Alternaria alternata, Penicillium spinulosum, and Mucor hiemalis were studied with respect to the action of some ecological factors: sucrose concentration (0, 0.2, 2, 10, and 100 g/l), temperature (4, 20, 25, and 30 degrees C), pH (3.5, 4.0, 5.0, 6.2, and 7.0), and cadmium concentration (0, 2, 10, and 100 mg/l). The spore germination rate and the viabilities of different mycelial fragments were found to reach their maxima at different values of the ecological factors studied. This finding suggests that the vegetative and asexual types of reproduction of microscopic fungi may have different ecological optima.

Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis

A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant ( Solanum tuberosum), nematode ( Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months, respectively. Both singly-inoculated soils and soils that had received mixed inoculants revealed, next to bands resulting from indigenous fungi, the expected bands in the DGGE profiles. The A. oligospora specific amplicon, by virtue of its unique migration in the denaturing gradient, was well detectable, whereas the T. harzianum specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiophene-containing petrol showed the progressive selection of specific fungal bands over time, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and analysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca, N. ochroleuca and Fusarium solani. Fungal isolates obtained from the treated soil on PDA plates were identified as Trichoderma sp., whereas those on Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp).

Effect of a Select Group of Seed Protectant Fungicides on Growth of Sclerotinia minor In Vitro and Its Recovery from Infested Peanut Seed.

Potato-dextrose agar containing 100 μg of streptomycin sulfate per milliliter of medium (SPDA) was amended to establish levels of 0, 2, 4, 6, 8, and 10 μg/ml of the fungicides thiophanate-methyl, carboxin, dicloran, captan, pentachloronitrobenzene (PCNB), or thiram. Fresh mycelial plugs, dry mycelial fragments, and sclerotia of Sclerotinia minor were placed onto the amended media, and mycelial growth and sclerotial germination were determined for 7 days. Thiophanate-methyl was the most effective chemical in inhibiting growth of S. minor, followed by PCNB and dicloran. Thiophanate-methyl was the only chemical that prevented germination of sclerotia of S. minor. Peanut seed naturally infested with S. minor was treated with the fungicides alone or in various combinations; control seed was treated with talcum powder. Seed was stored in polyethylene bags for 12 weeks at 24°C. To determine the incidence of viable S. minor in treated seed, seed was removed from bags, washed for 1 min in 0.2% unscented, liquid soap solution, rinsed twice in distilled water, and then dried for 15 min prior to plating on SPDA. The incidence of S. minor in talcum and thiophanate-methyl treated seed was 3.68 and 0.0%, respectively. Incidence of S. minor in seed treated with the other compounds ranged from 0.40 to 0.56%. Subsequent tests were performed using thiophanate-methyl and some of the above chemicals, in combinations or alone. These tests confirmed that thiophanate-methyl was the only compound that consistently reduced recovery of S. minor from infested seed. Germination of thiophanate-methyl treated seed equaled that of seed treated with talcum.

Agitation induced mycelial fragmentation of Aspergillus oryzae and Penicillium chrysogenum

Given the impact of mycelial morphology on fermentation performance, it is important to understand the factors that influence it, including agitation-induced fragmentation. The successful application of the energy dissipation/circulation function (EDC) to correlate fragmentation of Penicillium chrysogenum with agitation intensity and with different impeller types [5] has already been demonstrated. The EDC function takes into account the specific energy dissipation rate in the impeller swept volume and the frequency of mycelial circulation through that volume. In order to explore whether the EDC function can be used more generally to correlate fragmentation of different filamentous species, the present study extended the concept to agitation-induced, off-line fragmentation of Aspergillus oryzae grown in chemostat culture. The work shows that at EDC values off-line greater than that in the chemostat, fragmentation with different impellers can be correlated with the EDC. For EDC values less than those used in the chemostat, fragmentation did not occur. The earlier results of Jüsten et al. [5] with Penicillium chrysogenum are also reconsidered and found to behave similarly.

Pathogenicity of Sclerotinia sclerotiorum to Chrysanthemoides monilifera ssp. rotundata (Bitoubush) and Selected Species of the Coastal Flora in Eastern Australia

Abstract Chrysanthemoides monilifera spp. rotundata (bitoubush) is an invasive, noxious weed of coastal ecosystems in eastern Australia. Bushes infected by Sclerotinia sclerotiorum were sampled at nine locations along the New South Wales coast. All isolates of the fungus were pathogenic but there were significant differences in the virulence of isolates and in the resulting disease severity in bushes inoculated with fragmented mycelium. The most virulent isolate was chosen for pathogenicity testing on 45 species of plants in 21 families that are members of the common coastal native flora. Nine species developed lesions and Kennedia, Pandorea, Scaevola, and Westringia were the most susceptible genera, although their reaction to inoculation was variable and disease progression ceased on removal of plants from a humid environment. Only one plant (Carpobrotus sp.) died as a result of inoculation but no sclerotia developed on the diseased tissue of any native species. As no disease caused by S. sclerotiorum has been observed in the native flora, it is considered that this pathogen should be studied further as a biocontrol agent for bitoubush.

Evolution of Aspergillus niger and A. nidulans in glucose-limited chemostat cultures, as indicated by oscillations in the frequency of cycloheximide resistant and morphological mutants

Aspergillus niger and A. nidulans were grown separately in glucose-limited chemostat cultures on modified Vogel's medium. Periodic selection (the appearance of new mutant populations) in A. niger was determined by monitoring oscillations in the concentration of cycloheximide-resistant mycelial fragments in samples from the fermenter vessel. Using these data, the interval between the periodic selection of each new mutant population was 32 ± 6 generations (mean ± S.E.M.). Periodic selection in cycloheximide resistance in three morphologically distinct sub-populations of the A. niger culture averaged 36 ± 2, 31 ± 11 and 28 ± 5 generations and these values were not significantly different from that of the whole population (32 ± 6 generations). Also, it was possible to estimate the rate of evolution taking place in these cultures by monitoring oscillations in the frequency of morphological mutants. The intervals between the periodic selection of morphological mutant populations in A. niger and A. nidulans were 28 ± 3 and 22 ± 2 generations respectively.

Kinetics and manipulation of hyphal breakage and its effect on antibiotic production

Erythromycin production in 1.5 l continuous cultures of Saccharopolyspora erythraea, was correlated with the mean mycelial fragment size, which was influenced by culture agitation (750–1 500 rev./min marine/disk turbine impellors). Kinetic parameters for hyphal breakage were determined, and revealed that more highly branched mycelial fragments (hyphal growth unit ∼14 μm, compared to ∼21 μm) were more resistant to breakage. Branching rate depended on growth rate, the more highly branched mycelium being observed at the higher dilution rate. The breakage rate was inversely related to the mycelial fragment diameter at all dilution rates. Manipulating the size distribution of mycelial fragment by using an ultrasonic filter improved specific antibiotic productivity by 33%.

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

We studied the viability of fragmented myce- lium of Pisolithus tinctorius and Paxillus involutus en- trapped in calcium alginate gel to determine the effica- cy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28 7C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ra- tios of alginate and mycelium suspension to 0.7 M CaCl2. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus myce- lium was more than 90% viable when entrapped myce- lium was 10 to 50 days old, and Pisolithus tinctorius at- tained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days stor- age at 6 7C on dry filter paper. For gel-entrapped Pisol- ithus tinctorius mycelium, viability was highest when stored at 25 7C in 0.7 M CaCl2. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum.

Influence of humidity on production of pelleted fungal inoculum

One of the practical problems in scaling-up the production of fungal inocula for environmental applications is how to provide essential humidity for fungal growth. Pelleted solid substrate was used as a fungal biomass carrier. It was coated with alginate or agar hydrogels that contained mycelial fragments of the white-rot fungi Trametes versicolor or Irpex lacteus. To follow fungal growth and formation of mycelial coat over pelleted substrate, the fluorescein-diacetate hydrolysing activity (FDA) assay and visual inspection were used. Both fungi were able to overgrow the pelleted substrate in 5–6 days, at a relative humidity (RH) of 86.3% or higher. The enrichment of alginate hydrogel with nutrients or coating of pelleted substrate with more hydrophilic agar hydrogel enabled I. lacteus to overgrow the pellets at a lower RH of 83.6%. Fungal inocula produced at lower RH possessed lower final moisture contents and had greater mechanical strength. Conditioning of T. versicolor mycelial fragments, by a 3-h incubation in fresh growth medium, enhanced fungal growth over the pelleted substrate. A mathematical model was used to simulate and to explain moisture distribution in a hydrogel-coated pellet and the formation of mycelial coat, for various conditions of fungal inocula production.

Corticolous mites (Acari) as potential vectors of Cryphonectria parasitica (Murr.) barr hypovirulent strains

Specimens of four species of corticolous mites, Liebstadia humerata Sell. (Oribatida: Protobatidae), Scheloribates latipes (C. L. Koch) (Oribatida: Scheloribatidae), Thyreophagus corticalis (Mich.) (Acaridida: Acaridae), and Zygoribatula laubieri meridionalis Travé (Oribatida: Oribatulidae), were found in cankers, caused by Cryphonectria parasitica on chestnut trees, and were reared on laboratory cultures of the parasite. Monoxenic cultures were carried out using a hypovirulent (H) strain of the blight fungus. A group of mites was transferred from the H cultures to a met auxotrophic virulent (V) strain. Fecal pellets collected aseptically were used to inoculate agar plates. New C. parasitica cultures developed and white colonies with the morphological characters of the hypovirulent strain were obtained. Development of C. parasitica from mites indicated that the mycelial fragments were viable. The dsRNA presence in these cultures obtained from fecal pellets deposited by mites feeding from the blight agent mycelia was emphasized. Inoculation tests in the field confirmed that the white strains developed from the mite's fecal pellets were hypovirulent and suggested that these arthropods play a leading role in the natural spreading of hypovirulence in the chestnut stands.

Image analysis of the morphology of filamentous micro-organisms.

Overview Image analysis is a powerful tool for characterizing the gross and internal morphology of filamentous microorganisms. Methods are available for characterizing both dispersed and pelleted growth forms, and for identifying apices, vacuoles and degenerated regions of hyphae. The technique has also been applied to the morphological characterization of dimorphic yeasts. Such methods can be used to study the complex interactions between morphology, productivity and process conditions in submerged fermentations of filamentous micro-organisms, including the effects of changes in inocula, and to study the growth of such organisms on solid surfaces. Amongst other applications, image analysis has been used to relate fermentation broth rheology to morphology, to study mycelial fragmentation by impellers, and to investigate structural and biochemical differentiation of hyphae both on solid surfaces and in submerged fermentations. This review describes these image analysis methods and their actual and potential applications in the study of filamentous micro-organisms.

Growth and viability of mycelial fragments of white-rot fungi on some hydrogels

The viability of mycelial fragments of Trametes versicolor and Irpex lacteus and their growth on selected hydrogels are described. The size of mycelial fragments of the fungi did not significantly influence their viability. Alginate hydrogel films supported fungal growth better than agarose, carrageenan, chitosan and gelatin films, and had the highest mechanical strength but were less hydrophilic than the other hydrogels. All commercial alginates that were tested supported aseptic growth of fungal fragments without prior sterilization of the hydrogel solution. The viability of mycelial fragments in the hydrogel solutions was higher for some commercial alginates than that in laboratory grade alginate. The mechanical strength and hydrophilicity of hydrogels from alginate type Sobalg FD 155 and Meer HV were comparable to that of laboratory grade alginate. Sterilization and pH of the alginate hydrogel did not significantly influence the growth of T. versicolor mycelial fragments but affected the growth of I. lacteus. Concentrations of alginate in the range of 1‐2% in the hydrogel did not affect the growth of entrapped mycelial fragments of these fungi.

Measurement of autolysis in submerged batch cultures ofPenicillium chrysogenum

The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and beta-1, 3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in beta-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum.

An Australian isolate of Alternaria crassa shows potential as a mycoherbicide to control the weed Datura stramonium

An isolate of Altemaria crassa obtained from Datura stramonium was tested for its potential as a mycoherbicide in south-eastem Australia. Sporulation of A. crassa in culture was greatest under the following conditions: on V-8 juice agar, with a 12 h photoperiod of ‘Warm White’ light, with cultures arranged in a monolayer, at 25–30°C, at about 13 days after inoculation. Spores rather than mycelial fragments were required to kill seedlings of D. stramonium. Preliminary experiments indicated that a minimum dew period of 9 h and a spore concentration of 105/mL were required to kill cotyledonary-stage seedlings of D. stramonium. In host range studies in the glasshouse, A. crassa killed or severely reduced the growth of all Datura species tested and other solanaceous weeds. Also, A. crassa caused disease symptoms on some crops and ornamental species, including four tomato and two eggplant cultivars, Petunia × hybrida ‘Mardi Gras’ and a soybean cultivar. Microscopic examination of the infection process after inoculation showed that spores germinated, formed appressoria and penetrated equally well on different species and so resistance was intracellular. This local isolate of A. crassa is potentially an effective mycoherbicide for D. stramonium and other solanaceous weeds in south-eastem Australia.

Cephalosporin C Production by Cephalosporium acremonium: The Methionine Story

ABSTRACT: More than 40 years ago, it was reported that methionine markedly stimulated production of cephalosporin C by Cephalosporium acremonium. Over the years, many hypotheses were put forth to explain this phenomenon. The accumulating evidence strongly supported the concept that methionine stimulates by inducing enzymes of the biosynthetic pathway such as δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase. This mechanism has been strengthened by the finding that transcription of the genes encoding the above enzymes is markedly enhanced by growth with methionine. An effect of methionine in the fermentation unrelated to the titer stimulation is its contribution of the sulfur atom to the cephalosporin molecule. Methionine also stimulates mycelial fragmentation; the relationship between this effect on hyphal differentiation and the induction of the cephalosporin synthases remains to be elucidated.

Factors Affecting Germination and the Mode of Germination of Zygospores of Choanephora cucurbitarum

AbstractTreatment of zygospores of Choanephora cucurbitarum with KMnO4, NaClO or H2O2 effectively activated the spores and induced their germination. The optimum concentration of KMnO4 for activation of zygospores was 0.25 to 0.5%. Zygospores were not able to germinate in darkness even after activation by KMnO4. When zygospores from 40 to 50‐day‐old cultures were treated with 0.5% KMnO4 solution for 60 min before incubation on water agar at 24% under light, about 50% germinated in 10 days. KMnO4 treatment killed more than 99% of residual mycelial fragments, sporangiospores and sporangiola in the zygospore suspension. During germination disappearance of oil droplets in zygospores occurred prior to the cracking on zygospore wall. Both sporangial germination and mycelial germination were found. Moreover, sporangiole germination was observed for the first time.

Inoculum effects on the production of γ-linolenic acid by the shake culture of Cunninghamella echinulata CCRC 31840

The effects of using the fragmented mycelia as inoculum on the growth of and lipid and γ-linolenic acid (GLA) production by the sporulation-deficient fungal strain Cunninghamella echinulata CCRC 31840 were examined in shake cultures. In a 5-day culture sown with 1.6% inoculum, the biomass increased 51.5%. Its lipid content increased 69.0% when the floc massiveness of the inoculum was increased from 27,000 to 81,000 colony forming units per milliliter (CFU ml −1). This in turn resulted in a synergistic increase of GLA yield from 358 to 860 mg l −1 whereas the GLA content of the lipid was virtually unaffected (11.2–13.4%). The maximum GLA yield of 1,349 mg l −1 was obtained in a 5-day culture when the production medium was pitched at the 4% level with an inoculum preparation in which 1.0 g of mycelia have been fragmented to a floc massiveness of 83,000 CFU ml −1 by vortexing for 4 min.

Aging of Penicillium chrysogenum cultures under carbon starvation: II: protease andN‐acetyl‐b‐D‐hexosaminidase production

In aging carbon‐depleted cultures of Penicillium chrysogenum the fragmentation and autolysis of old mycelia were coupled with the production of high levels of extracellular protease and N‐acetyl‐b‐D‐hexosaminidase; this could be stopped by the addition of an extra dose of glucose at any incubation time tested, but was not affected by endogenous NH3. After the addition of glucose, intracellular enzyme accumulation was observed only for the N‐acetyl‐b‐D‐hexosaminidase, and the concomitant decrease in both the extracellular protease and N‐acetyl‐b‐D‐hexosaminidase activities was not caused by the action of extracellular proteases. The physiological function of the N‐acetyl‐b‐D‐hexosaminidase remains to be elucidated because the P. chrysogenum culture studied did not utilize N‐acetyl‐D‐glucosamine as a carbon source. However, the proteases (mainly serine and, to a much smaller extent, metalloproteases) might provide surviving hyphal fragments with sufficient amino acids to maintain the cryptic growth observed in aging carbon‐depleted cultures.

Allergic potential of someAspergilli on saw mill workers in Lucknow, India

Fifty fungal types were isolated from the indoor atmosphere of saw mills by exposing Petri plates containing Czapek-dox Agar, Potato-dextrose Agar and Sabouraud Agar media for 5 min. The fungal flora of the outdoor surroundings was also studied for comparison. Species ofAspergilli dominated in the saw mills, being represented by 16 species including one ascosporic form. Other fungi were species ofCladosporium, Alternaria, Curvularia, Penicillium, Fusarium, etc. Variations in the fungal population in different months were also observed. Fungal spores recovered using the Rotorod Sampler wereAlternaria, Curvalaria lunata, Curvularia tetramera, Cladosporium, Dreschslera sp.,Epicoccum sp.,Pithomyes sp.,Nigrospora, Stemphylium sp. andTorula sp. Mycelial fragments and unidentifiable spores were also seen in abundance. Varying allergic responses of patients were also recorded by testing intradermally, the antigens of nineAspergilli, vizAspergillus flavus, A. fumigatus, A. japonicus, A. melleus, A. nidulans, A. niger, A. niveus, A. tammarii and A. terreus.