Hyphal Fragments Research Articles

Hyphal vocuolation and fragmentation inpenicillium chrysogenum

A link between vacuolation and fragmentation of Penicillium chrysogenum mycelia in stirred tank submerged fermentations is reported. Quantitative information on vocuolation and morphology was obtained by image analysis. In fed-batch fermentations the coincidence of the events of rapid vacuolation and the fall of the mean total and main hyphal lengths suggests that hyphal fragmentation is not necessarily due to "shear" alone. The physiological state of the hyphae, characterized by the proportions of vaccuoles, was found to have a significant influence on the breakage of mycelial hyphae, It was found that the fragmentation was greater when the hyphae became heavily vacuolated following nutrient limitation in the culture, i.e., during the switch from the rapid growth to the production phase.

Establishment and Meloidogyne-capture of an adhesive hyphaeforming isolate of the fungus Arthrobotrys oligospora in soil

In situ observations of an isolate of Arthrobotyrs oligospora (CBS 289.82) in sterilized and untreated soil revealed attachment of second-stage juveniles of the root-knot nematode Meloidogyne hapla to hyphae, development of rings around the nematode body and nematode colonization by trophic hyphae. Abundant growth of rings from infected nematodes was never observed. Establishment and nematode capture by this isolate was compared with those of several other fungi from the Dactylaria-complex in a simple soil microcosm system. Application of hyphal fragments of A. oligospora (CBS 289.82), about 30 mm g −1 oven-dry soil, resulted in hyphal lengths of 100–170 m g −1 oven-dry soil, reduction of 90% of living juveniles of M. hapla within 1 day and extermination of the nematodes after 10 days. Arthrobotrys oligospora (CBS 289.82) performed far better than any of the other fungi. Establishment in non-sterilized soil was not as good, but still total hyphal length of about 10 g −1 oven-dry soil resulted in 70% reduction of living nematodes at Day 10. At 13°C results were similar to those at 20°C. The results support the conclusion that adhesive hyphae-forming fungi may be useful for the control of nematodes in soil.

LATENT DECAY FUNGI: THE HIDDEN FOE?

Summary In the sapwood of branches and trunks of angiosperm trees, extensive (several, sometimes many metres) columns af decay, occupied by a single fungal individual frequently develop rapidly (often in less than one growing season), in the absence of major wounds. Such rapid and extensive colonization is unlikely to result from mycelial extension from a single focus. However, the patterns can be explained by the presence of latent fungus propagules (such as yeasts, hyphal fragments or spores) distributed widely but sparsely in the sapwood, but which do not develop when sapwood is functional in water conduction. The widespread presence of such latent fungi has been demonstrated in the laboratory where lengths of what had been healthy twigs, branches and trunks of ash, beech, oak and hazel were dried under controlled conditions. There is also considerable circumstantial evidence that development of these latent propagules into mycelia, which cause decay, occurs in the field when sapwood begins to dry, as ...

Refined Procedures for Inoculating Wheat Seedlings withPyrenophora tritici-repentisand Rating Their Reactions

Systems for rating reaction of wheat to infection by Pyrenophora tritici-repentis, the tan spot pathogen, should be informative and reliable so that resistant cultivars can be efficiently developed. Therefore, the effects of propagule type, inoculum dosage, and spatial interaction among lesions on infection phenotype, infections per unit area, and percent disease severity were studied. Conidiophore inoculation of seedlings of wheat line ND 495, which is susceptible to tan spot, produced a lower infection phenotype, fewer infection sites per square centimeter, and less severe damage than conidia at equivalent dosages. Hyphal fragments were rarely infectious under a 24-hr postinoculation wet period. The relative reaction of the resistant wheat cultivar Eric to the three types of inoculum was similar to that of ND 495 [...]

Histopathology Of Sinonasal Fungal Disease

1. To increase diagnostic yield, the otolaryngologist should convey suspicions of mycotic sinus disease to the pathologist. 2. Fungal cultures are a necessary adjunct to histopathologic identification. 3. Any sinonasal chronic inflammatory or granulomatous sinus process, or allergic process, may have a fungal etiology, even with negative GMS stains. 4. The diagnosis of sinonasal "inflammatory pseudotumor" should be accepted with some reservation, as it may represent an unrecognized sinonasal mycotic process. 5. Aspergillus spp. may be definitively identified histologically in sinonasal mycetomas and in invasive disease. When only fragments of thin septate hyphae, are found, one cannot assume they always belong to Aspergillus. 6. One must not assume that all wide "pleomorphic" hyphal fragments belong to Rhizopus or, for that matter, Zygomycetes. 7. Candida, Pseudoallescheria, Rhinosporidium and Cryptococcus are rare sinonasal pathogens that may also be positively identified morphologically.

Synchronous Sporulation of Ascosphaera Proliperda

A minimal medium containing glucose, phosphate-buffered saline, and (NH4)2S04, CaCl2, MgS04, FeS04, ZnS04, and agar was developed to insure the production of large number of synchronous ascocysts of Ascosphaera proliperda with minimal vegetative growth. This fungus is a larval pathogen of the alfalfa leaf cutting bee, Megachile rotundata. The synchronous production of ascocysts was essential for the isolation of two proteins associated with sexual reproduction of this fungus. To develop this medium, pH, carbon and nitrogen sources, and trace elements were studied for their effects on the differentiation of ascocysts. Over the pH range 5.4-8, pH 6 was the most suitable for production of synchronous ascocysts in abundance. Size of colonies grown on media containing galactose, sucrose, or glucose did not vary significantly; however, more ascocysts were produced with medium containing either sucrose or glucose. Only (NH4)2S04 at concentration of 0.25% was suitable as a nitrogen source. Other sources such as (NH4)2C03, NH4C1, and NH4N03 were not suitable for fungal growth or production of ascocysts. Medium lacking trace elements (B, CI, Cu, Fe, Mg, Mn, Mo, S, Zn) did not support fungal growth or ascocyst formation; only Fe, Mg, S and Zn were necessary for sporulation. To maximize synchronous contact between the two mating types, a mixture of hyphal fragments of both mating types was used as an inoculum in the tested media.

Synchronous Sporulation of Ascosphaera proliperda

A minimal medium containing glucose, phosphate-buffered saline, and (NH4)2SO4, CaCl2, MgSO4, FeSO4, ZnSO4, and agar was developed to insure the production of large number of synchronous ascocysts of Ascosphaera proliperda with minimal vegetative growth. This fungus is a larval pathogen of the alfalfa leaf cutting bee, Megachile rotundata. The synchronous production of ascocysts was essential for the isolation of two proteins associated with sexual reproduction of this fungus. To develop this medium, pH, carbon and nitrogen sources, and trace elements were studied for their effects on the differentiation of ascocysts. Over the pH range 5.4-8, pH 6 was the most suitable for production of synchronous ascocysts in abundance. Size of colonies grown on media containing galactose, sucrose, or glucose did not vary significantly; however, more ascocysts were produced with medium containing either sucrose or glucose. Only (NH4)2SO4 at concentration of 0.25% was suitable as a nitrogen source. Other sources such as (NH4)2CO3, NH4Cl, and NH4NO3 were not suitable for fungal growth or production of ascocysts. Medium lacking trace elements (B, Cl, Cu, Fe, Mg, Mn, Mo, S, Zn) did not support fungal growth or ascocyst formation; only Fe, Mg, S and Zn were necessary for sporulation. To maximize synchronous contact between the two mating types, a mixture of hyphal fragments of both mating types was used as an inoculum in the tested media.

Functional and evolutionary implications of a survey of various actinomycetes for homologues of two Streptomyces coelicolor sporulation genes

In Streptomyces coelicolor A3(2) the whiB and whiG genes are essential for sporulation, their deduced products being a possible transcriptional activator and an RNA polymerase sigma factor, respectively. In a survey of DNA from diverse actinomycetes by Southern blotting, all samples tested hybridized with whiB, but only those representing genera capable of producing sporulating aerial mycelium hybridized with whiG. It is postulated that whiB may play a more intimate role in hyphal fragmentation processes (including sporulation) than whiG. The whiB and whiG homologues (whiB-Stv and whiG-Stv) of Streptoverticillium griseocarneum were cloned and sequenced, and subjected to functional tests in S. coelicolor whiB and whiG mutants. The genes were closely similar, but not identical, to their S. coelicolor counterparts at the DNA and deduced protein levels, and both Stv. griseocarnum gene products could function well in S. coelicolor. However, studies with hybrid transcription units suggested that the promoter region of whiB-Stv is somewhat inefficient in S. coelicolor.

Characterization of the parasexual cycle in the eyespot fungus, Pseudocercosporella herpotrichoides

Parasexual recombination in the eyespot fungus, Pseudocercosporella herpotrichoides , is demonstrated using complementary auxotrophic mutants derived from the W-type isolate 22–20. The parasexual cycle follows a typical pattern consisting of a mosaic heterokaryon phase, a relatively stable diploid phase and various putative aneuploid stages leading to recombinant haploid formation. The heterokaryon can be resolved into its component homokaryons either by isolating single hyphal tips or from single conidia, which remain homokaryotic. The non-sporing diploids can be isolated from random plating of spores and hyphal fragments taken from heterokaryotic colonies, or by regenerating protoplasts isolated from heterokaryotic mycelia. An efficient method for obtaining diploids was developed, which exploits the particularly slow growth rate of heterokaryons formed between complementary histidine auxotrophs ( Hisl, Hien, Hi ). Diploid mycelium grows out as faster growing sectors from such heterokaryons. Segregation yielding recombinant haploids followed exposure to 5-fluorouracil. Various putative aneuploid stages were observed, typified by their very slow growth rate and instability. Spontaneous outgrowths yielding stable recombinant types were recovered from these unstable colonies following maceration of mycelium and plating on a non-selective, complete medium. A cross was performed to investigate the inheritance of benzimidazole and N -phenylcarbamate fungicide resistance. After retesting of resistance phenotypes, cosegregation of carbendazim resistance and increased sensitivity to the phenylcarbamate MDPC was confirmed in most cases. One exception involved a putative recombinant in which sensitivity to both carbendazim and the phenylcarbamate was combined. This recombinant was obtained after exposure of the original diploid to 5-fluorouracil, known to stimulate mitotic crossing-over in other fungi. However the possibility remains that it represents a chance spontaneous mutation either in the resistance gene or at some other locus.

Soil disturbance and infection of Trifolium repens roots by vesicular-arbuscular mycorrhizal fungi

Removal and storage of the surface layers of soil is known to decrease the infectivity of vesicular-arbuscular mycorrhizal (VAM) fungi. Previous studies have mostly examined the effects of profound soil disturbance on the infectivity of VAM fungi. This study examined the effects of increasing degrees of topsoil disturbance on the infectivity of VAM fungi in two sites on sandstone soils in southeastern Australia. Intact soil blocks (20×20×15 cm) were taken from each of the two sites. Increasing degrees of topsoil disturbance were achieved by cutting the blocks longitudinally into four (dist. 1), nine (dist. 2), and 25 (dist. 3) equal portions. Seeds of Trifolium repens L. were sown into the blocks and harvested 14, 21, 28, 35 and 42 days after sowing. At each sampling date, total root length, root length colonised by VAM fungi and shoot dry mass were measured. VAM colonisation had commenced by 14 days in the roots of seedlings grown in intact, dist. 1, and dist. 2 soil blocks. The initiation of VAM colonisation was delayed by up to 6 weeks for seedlings grown in the dist. 3 soil blocks. The low (i.e. dist. 1) and intermediate (i.e. dist. 2) degrees of soil disturbance did not cause a delay in the initiation of VAM, bud did significantly reduce the proportion of root length colonised by VAM fungi after 21 days. After 21 days, shoot dry mass was significantly less in the seedlings grown in the dist. 3 soil blocks though not in the low and intermediate disturbance treatments. It is concluded that the most severe experimental disturbance probably disturbed the external hyphal network and root fragments (containing hyphae and vesicles), which in turn temporarily reduced the infective potential of the fungus to zero. The observed delay in the initiation of VAM in the most disturbed blocks can, therefore, be explained by the time required for hyphae to grow from other propagules in the soil which survived the disturbance event.

Mycelial fragment size distribution: an analysis based on fractal geometry

The use of white-rot fungi for biodegradative research and developmental application requires seeding with a suitable inoculum. This paper presents a new method for the quantitative analysis of a mycelial inoculum composed of homogenized hyphal fragments. Our method is premised on a power-law behaviour between frequency and the size of these mycelial fragments. It is shown that the fragment distribution profile can be determined by regression to give the fractal fragmentation dimension, D. The influence of homogenizer speed was also investigated over a range from 8333 rpm to 25 000 rpm, which corresponds to a shear rate range of 13.9 × 103 to 41.7 × 103 s−1. The highest D value was shown at a shear rate of 27.8 × 103 s−1 for 30 s, implying greatest homogeneity in the size distribution function over the measured range (0–500 μm2). As shear force and duration increases (up to a threshold value) the production of small fragments is facilitated with a corresponding decrease in the D value. The slope relation express the fragment diversity whereas the reciprocal fractal valie characterizes the distribution size probability. Image-analysis methodology is described and the implications of a fractal description of a mycelial inoculum are also considered.

Accumulation of photoassimilate by Peronospora viciae (Berk.) Casp. and leaves of Pisum sativum L.: evidence for nutrient uptake via intercellular hyphae

SUMMARYAutoradiography showed that accumulation of label by hyphae of Peronospora viciae (Berk.) Casp., in leaflets of Pisum sativum L. exposed to 14CO2, was up to 4.5 times greater in the susceptible cv. Krupp Pelushka than the resistant cv. Early Onward. Concentrations of label were similar in apoplastic solutions from both cvs, although a greater volume was available in Early Onward. Uninfected Krupp Pelushka assimilated more label from 14CO2 than Early Onward; para‐chloro‐mercuribenzene sulphonic acid (PCMBS) treatment and infection reduced assimilation by Krupp Pelushka but not Early Onward. Infected leaf tissue from Early Onward assimilated more label than Krupp Pelushka from [14C]sucrose supplied to the apoplast. Transient treatment with PCMBS reduced assimilation of [14C]sucrose into infected plant tissue to 30 and 61 % of untreated levels, in Early Onward and Krupp Pelushka respectively. In contrast, autoradiography of hyphal fragments showed that the fungus accumulated 1.4 times more label from [14C]sucrose than without PCMBS. The different nutrient status of leaves of Early Onward and Krupp Pelushka, and evidence that label from sucrose is accumulated by intercellular hyphae directly from the apoplast, are discussed in relation to the role of haustoria and mechanisms of compatibility.

Spectrophotometric determination of mycelial biomass

The optical density (450 nm) of samples of homogenized fungal biomass correlated linearly with the dry weight of the biomass in the samples. As shown for broths of the filamentous microfungus Neurospora sitophila, the sensitivity of the technique depended on the extent of fragmentation of fungal hyphae during homogenization: increased fragmentation increased sensitivity. The method applied during all phases of growth, was as accurate as the conventional dry weight technique and permitted rapid and simple measurement of biomass concentration.

A membrane technique for producing protoplasts ofCryphonectria parasitica

A method for the formation and regeneration of protoplasts of several strains of the chestnut blight fungus,Cryphonectria parasitica, is presented. The procedure utillizes cellophane membranes for growth and employs centrifugation for separation of protoplasts from hyphal fragments. Yields averaged 8.04×106 protoplasts per membrane. Regeneration frequencies were 40–50% with a soft-agar overlay. These protoplasts are suitable for use in experiments designed to determine the role of dsRNA in hypovirulence ofC. parasitica.

Enhanced Production ofPyrenophora tritici-repentisConidial Suspensions

A technique that yields conidial suspensions of Pyrenophora tritici-repentis nearly free of other propagules involves growing the fungus in potato-dextrose broth, followed by removal, comminution, and inoculation into liquid clarified V8 juice agar (CV8). After 48 hr of growth in the dark, the fungus is exposed to alternating periods of light and dark that stimulate the production of conidiophores and conidia in a lawn covering the entire surface of the colony. In two tests using 24 isolates of P. tritici-repentis, inoculum suspensions were obtained with an average of 1,386 and 1,692 propagules (conidia, conidiophores, and hyphal fragments) per milliliter, in which 92 and 100%, respectively, of the propagules were conidia

Influence ofPseudomonas fluorescenson Hyphal Growth and Biocontrol Activity ofTrichoderma harzianumin the Spermosphere and Rhizosphere of Pea

Trichoderma harzianum isolate ThzID1 was grown in liquid culture, was formulated with alginate and polyethylene glycol 8000, and was milled into fine granules (average diameter 500 μm). Granules contained chlamydospores, conidia, and hyphal fragments. Viability of the encapsulated fungus remained high for at least 6 mo when stored at 5 C (i.e., >90% of the granules produced hyphal growth when incubated on agar); viability was reduced significantly when granules were stored at 22 C. Application of the granular formulation of T. harzianum to pea seeds reduced root rot by Aphanomyces euteiches f. sp. pisi in growth-chamber experiments and also increased plant top weights compared to noncoated seeds []

The propagules of vesicular-arbuscular mycorrhizal (VAM) fungi capable of initiating VAM infection after topsoil disturbance

The removal and storage of topsoil decreases the infectivity of vesicular-arbuscular mycorrhizal (VAM) fungi. The propagules of VAM fungi include spores, root fragments containing hyphae and vesicles, and soil hyphae. The viability of each type of propagule after disturbance will determine the initiation of VAM associations with plants recolonizing the disturbed site. This study aimed to examine which of the propagules of VAM fungi are capable of initiating VAM infection after soil disturbance. Soil from an open woodland site of low soil fertility, in southeastern Australia was wetsieved through a tier of three sieves (1 mm, 250 μm and 106 μm), and the following fractions were extracted: (i) root fragments, (ii) fungal hyphae, and (iii) VAM spores. Each fraction was tested to determine its potential to initiate VAM. Hyphae of VAM fungi grew from root fragments within 14 days. The VAM spore fraction initiated VAM infection after 28 days. VAM hyphal fragments did not produce any VAM infection even after 42 days.

Entomophthoromycosis due to Conidiobolus.

Entomophthoromycosis due to Conidiobolus coronatus is a granulomatous infection characterized by lesions that originate in the inferior turbinate, spread through ostia and foramina to involve the facial and subcutaneous tissues and paranasal sinuses. The majority of the cases have been described from areas of tropical rainforest in West Africa, agricultural and outdoor workers (aged 20-60 years) being the ones most frequently affected. The fungus is common in soil and decaying vegetation. Infection probably occurs by implantation of the spores of the fungus in nasal mucosa. C. incongruus is a rare agent of the disease, so far known only from two cases with lesions involving the pericardium, mediastinum, lungs, liver, oesophagus and jejunum. C. coronatus is known to cause a clinically similar disease in horses, mules, a dolphin and a chimpanzee. A characteristic histological feature is the presence of thin-walled, broad, often septate hyphae or hyphal fragments with a thick eosinophilic sheath, frequently phagocytosed within giant cells. The fungus is known to produce in vitro several enzymes, e.g., elastase, esterase, collagenase and lipase, which have a possible role in pathogenicity. A concentrated brain heart infusion culture filtrate antigen is useful for immunodiagnosis. Several drugs e.g., potassium iodide, cotrimoxazole, amphotericin B, ketoconazole and itraconazole have been tried with varying success. Investigations on the immunology of disease and the role of proteases and lipases in the pathogenesis of infection is an important area of further research.

The role of mycorrhizas in the regeneration of some Malaysian forest trees

An investigation was made into the availability of mycorrhizal inoculum and the response of tree seedlings to mycorrhizal infection in West Malaysian forests. Spores of vesicular arbuscular (VA) mycorrhizal fungi in the soil were reduced by 25% after selective logging and by 75% after heavy logging, VA infection in the roots of plants persisting on, or colonizing, a heavily logged site was reduced by up to 75% . The most probable number (MPN) of VA propagules in sieved soil was up to ten times greater than spore density, but was also greatly reduced by heavy logging. This resulted in reduced infectivity of soil from the heavily logged site, as demonstrated by reduced VA infection of bioassay plants. The infectivity of soil declined following sun drying, but sun-dried soil devoid of vegetation retained some infectivity even after 12 months storage. Overall the data suggest that root and hyphal fragments are more important than spores as inoculum in disturbed forest, and that in undisturbed forest living roots and hyphae are likely to be important sources of infection. In a pot experiment, shoot growth of two test species, Albizia falcataria (L.) Becker and Parkia speciosa Hassk. responded more to VA mycorrhizal infection than to P fertilization over the range 0-6 g triple superphosphate per 8 kg of soil. The response to inoculation with a cocktail of ‘introduced’ VA fungi propagated in pot cultures was greater than the response to inoculation with ‘indigenous’ fungi propagated in pot cultures from roots and soil collected in undisturbed forests. Another test species, Intsia palembanica Miq., also responded better to mycorrhizal infection than to P fertilization, and better to VA mycorrhizal infection than to ectomycorrhizal infection. Intsia palembanica seedlings growing around mature dipterocarps quickly became ectomycorrhizal, suggesting that at least some ectomycorrhizal fungi infect both dipterocarps and Intsia. Shorea leprosula Miq. seedlings growing naturally in the forest had ectomycorrhizas 20 days after germination, i.e. before they had true leaves, and within 7 months supported up to 11 different ectomycorrhizal fungi. However, seedlings isolated from contact with the roots of mature Shorea trees remained uninfected in the held for up to 6 months. This shows the importance of contact with living ectomycorrhizal roots for early infection of dipterocarp seedlings, a point which should be recognized in logging operations and forest regeneration programmes.

Fungal microcolonies on indoor surfaces — an explanation for the base-level fungal spore counts in indoor air

In the subarctic winter, fungal spores are found in indoor air even when outdoor spore levels are very low. The results of this study support an explanation that some indoor airborne fungal spores are derived from unnoticeable fungal microcolonies, which may develop on temporarily wet surfaces. Laboratory experiments on Penicillium verrucosum indicated that the fungus germinated on new wallpaper very quickly (about half an hour) under moist conditions. Hyphal growth and sporulation of the fungus on moist wallpaper occured within one day of incubation. In gravity-settling tape samples from occasionally wet surfaces in a suburban home, large spore aggregates, hyphal fragments with some spores and spores in the germination stage were found, indicating fungal growth. These experiments showed that fungal microcolonies can develop within a week on occasionally wet indoor surfaces.