Basidiospore Germination Research Articles

Inhibition of basidiospore germination by western redcedar heartwood extractives

Understanding the relationship between extractives and decay resistance in western red cedar (WRC) is essential for breeding durable planting stock. To date, most such work has focused on resistance to mycelial attack. However, this is potentially misleading since WRC is largely used in above-ground applications where basidiospore germination is the primary way in which the wood is initially colonized. Efficacy against basidiospores is likely a critical factor affecting wood’s decay resistance in above-ground applications. In this study the effect of selected extractives on basidiospore germination was evaluated. Partially-extracted WRC veneer samples, and spruce samples treated with beta-thujaplicin, thujic acid, or plicatic acid were used. Beta-thujaplicin and thujic acid were associated with significantly reduced rates of Gloeophyllum sepiarium, Fomitopsis palustris, and Dichomitus squalens basidiospore germination. Plicatic acid was not associated with any effect on basidiospore germination. Planting stock that generates heartwood with high concentrations of thujaplicins and thujic acid should be selected to yield wood that will be resistant to basidiospore germination.

Rhizopogon roseolus basidiospore germination decreases as the fruiting bodies mature and the spore wall becomes more complex

Rhizopogon roseolus (Corda) Th. Fr. is a basidiomycete truffle that is considered edible. Its gleba changes color from white to beige to brown as it matures. Although ultrastructural changes in the spore wall have been linked to the maturation of the fruiting body, little is known regarding the relationship between spore germination success and the ultrastructure of the spore wall. We examined spore germination on agar plates and analyzed the spore wall ultrastructure using transmission electron microscopy (TEM). Fruiting bodies, collected from a pine forest, were classified into three developmental stages based on gleba color, and the germination success was evaluated at each stage. Variability in the spore germination rate was observed between individual fruiting bodies. The peak germination rate was recorded for the spores from the fruiting bodies in the beige glebal stage, and the rate was lower in the brown glebal stage. When spore wall structures were studied using TEM, the spore wall was found to be multilayered upon maturation of the fruiting body. The spores of the beige glebal stage showed three types of spore walls, namely two-layered, three-layered, and four-layered spore walls. On the other hand, the spores of the brown glebal stage showed predominantly four-layered spore walls. These results indicate that spore germination of R. roseolus decreases as the fruiting body matures and the spore wall becomes more complex.

EFFECTS OF LIGHT WAVELENGTHS AND COHERENCE ON BASIDIOSPORES GERMINATION

The effects of light wavelengths and coherence on basidiospore germination of Agaricus bisporus, Flammulina velutipes, Ganoderma applanatum, Ganoderma lucidum, Hericium erinaceus, Lentinus edodes and Pleurotus ostreatus have been studied. Short-term low-intensity irradiation by coherent (laser) light wavelength 488.0 nm and 632.8 nm at doses 45 and 230 mJ/cm2 has significantly increased the number of germinated basidiospores. It has established that there are differences in the photosensitivity not only between species but also between strains. Spores irradiation by 514.5 nm light has been either neutral or inhibitory. A comparative analysis of basidiospores sensitivity to laser and LED light has also been conducted. To stimulate germination of basidiospores and growth of monokaryons the most suitable solution was to use red coherent and incoherent light of 632.8 nm and 660,0 nm for A. bisporus, G. applanatum and P. ostreatus, red and blue coherent light of 632.8 nm and 488,0 nm for F. velutipes, and both red and blue laser and LED light G. lucidum and H. erinaceus and for L. edodes. No essential difference of a continuous wave mode and intermittent mode light effect at the same doses and wavelength on spore germination were revealed. Light influence has reduced germination time and formation of aerial mycelium on agar medium as compared to the original value and increased the growth rate of monosporous isolates. Characterization of basidiospores photosensitivity and development of environmentally friendly stimulating methods of their germination is important for creating highly effective technologies of mushrooms selection and cultivation.

Fungicide Impact on in vitro Germination of Basidiospores of Puccinia horiana, the Causal Agent of Chrysanthemum White Rust

Puccinia horiana is an actionable pathogen that, upon diagnosis, triggers an eradication protocol combining destruction of symptomatic chrysanthemums and a strict fungicide regime for asymptomatic plants. Symptoms typically appear during the fall as growers prepare to ship their crops. To expand the list of effective fungicides and develop fungicide sensitivity baselines, we screened in vitro germination of P. horiana basidiospores in 0.05% water agar solution amended with varying concentrations of 14 fungicides: azoxystrobin, boscalid + pyraclostrobin, fluoxastrobin, mancozeb, mandestrobin, metconazole, myclobutanil, propiconazole, tebuconazole, triadimefon, trifloxystrobin, trifloxystrobin + triadimefon, and triticonazole. Leaves with pustules ready to sporulate were affixed to petri plate lids over bases containing fungicide-amended agar. After 2 days in the dark, percent basidiospore germination was assessed. Concentrations required for 50% germination (EC50) grouped according to fungicide mode of action. Benzimidazoles exhibited EC50 values ranging from 9 to 244 ppm, while strobilurins ranged from 2 to 27 ppb. Mancozeb exhibited an EC50 of 7 ppm, and chlorothalonil was 205 ppb. Combinations of strobilurins with other modes of action exhibited EC50 values in the same range as the strobilurins. These data provide a baseline for monitoring resistance development to P. horiana over time. Accepted for publication 15 January 2015. Published 7 April 2015.

The infection process of Moniliophthora perniciosa in cacao

The development of the basidiomycete Moniliophthora perniciosa in resistant and susceptible Theobroma cacao genotypes was analysed. The infection process leading to broom formation in shoot apexes was characterized by studying the kinetics of basidiospore germination, mode of penetration and colonization of the pathogen. Both resistant and susceptible cacao genotypes were inoculated with M. perniciosa and kept in the greenhouse for 90 days, explants were collected, treated for histological studies and meristematic tissues were observed by electron and light microscopy. Variation in the kinetics of germination between the cacao genotypes was detected 4 h after inoculation. The fungal penetration occurred through the star‐shaped trichome base, natural openings on the cuticular surface and stomata. Host responses between genotypes were found to be different. Compared with non‐infected plants, the swelling of all the stem tissues was evident at 60 days after inoculation. In the susceptible genotype, typical symptoms developed and fungal colonization was more intense than in resistant genotypes, which showed little or no fungal colonization. The investigations reported herein provide an important step in understanding the pattern of pre‐ and post‐penetration events of M. perniciosa in cacao genotypes with different levels of resistance to this disease.

Evidence for genetic incompatibilities associated with post-zygotic reproductive isolation in the human fungal pathogen Cryptococcus neoformans.

Hybridization is a potent mechanism for generating unique strains with broad host ranges and increased virulence in fungal pathogens. In the opportunistic basidiomycete pathogen Cryptococcus neoformans, intervarietal hybrids are commonly found infecting patients. The two parental varieties C. neoformans var. grubii and C. neoformans var. neoformans mate readily under laboratory conditions, but the hybrid basidiospores have germination rates about four times lower than those from intravarietal crosses. Here, we used microdissection to collect basidiospores from a hybrid cross and analysed the genotypes of germinated basidiospores to identify potentially antagonistic allelic combinations between loci that impact basidiospore germination. Our analyses showed clear evidence for Bateson-Dobzhansky-Muller (BDM) incompatibility affecting basidiospore viability. Antagonistic combinations of alleles from both two loci and three loci were found. Interestingly, most of the hybrid progeny showed segregation distortion in favour of the alleles from var. neoformans, consistent with large-scale epistatic interactions among loci affecting basidiospore viability. Our study presents the first evidence of BDM incompatibility between nuclear genes affecting post-zygotic reproductive isolation in this model basidiomycete yeast.

Functional Diversification of Cerato-Platanins in Moniliophthora perniciosa as Seen by Differential Expression and Protein Function Specialization

Cerato-platanins (CP) are small, cysteine-rich fungal-secreted proteins involved in the various stages of the host-fungus interaction process, acting as phytotoxins, elicitors, and allergens. We identified 12 CP genes (MpCP1 to MpCP12) in the genome of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao, and showed that they present distinct expression profiles throughout fungal development and infection. We determined the X-ray crystal structures of MpCP1, MpCP2, MpCP3, and MpCP5, representative of different branches of a phylogenetic tree and expressed at different stages of the disease. Structure-based biochemistry, in combination with nuclear magnetic resonance and mass spectrometry, allowed us to define specialized capabilities regarding self-assembling and the direct binding to chitin and N-acetylglucosamine (NAG) tetramers, a fungal cell wall building block, and to map a previously unknown binding region in MpCP5. Moreover, fibers of MpCP2 were shown to act as expansin and facilitate basidiospore germination whereas soluble MpCP5 blocked NAG6-induced defense response. The correlation between these roles, the fungus life cycle, and its tug-of-war interaction with cacao plants is discussed.

Paratritirachium curvibasidium, a new heat-resistant basidiomycete from flare pit soils in Alberta, Canada

Paratritirachium is a recently described, monotypic, basidiomycete genus in the Pucciniomycotina with one described asexual species, P. cylindroconium. Three strains of an undescribed species of Paratritirachium were isolated from flare pit soils from Alberta, Canada, using a heat treatment method. Both asexual and sexual propagules were produced in culture. Asexual reproduction was characterized by unbranched or sparingly branched conidiophores and sympodial, slightly vesicular conidiogenous cells producing aseptate conidia. Distinctive curved basidia were commonly produced in culture, yielding two pale brown, thick-walled and oval basidiospores. The basidiospores were heat-resistant and germinated only when exposed to heat. Phylogenetic analyses using the internal transcribed spacer (ITS) region and the combined small and large ribosomal subunits confirmed that these isolates are a new species of Paratritirachium, described in this study as P. curvibasidium, and that Paratritirachium should be classified in the class Tritirachiomycetes, order Tritirachiales, and family Tritirachiaceae. Transmission electron microscopy revealed simple, uniperforate septa, similar to those of a Tritirachium species, supporting the placement of Paratritirachium in the class Tritirachiomycetes. This study provides the first description of sexual reproduction in the Tritirachiomycetes and the first report of basidiospore germination after heat treatment at a high temperature (75 °C).

The activity of phosphoinositide-specific phospholipase C is required for vegetative growth and cell wall regeneration in Coprinopsis cinerea

Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.

Optimal growth conditions for basidiospore germination and morphogenesis of Philippine wild strain of Lentinus tigrinus (Bull.) Fr.

Optimal growth conditions for basidiospore germination and morphogenesis of Philippine wild strain of Lentinus tigrinus (Bull.) Fr.

Effects of environmental factors on basidiospore germination of ammonia fungi Coprinopsis spp. collected from different geographical areas

Effects of pH, NH4-N, and temperature on basidiospore germination in Coprinopsis austrophlyctidospora from New Zealand, C. phlyctidospora from Japan, C. aff. rugosobispora from Canada, and C. echinospora from Canada were investigated. The Coprinopsis spp. required the presence of ammonium-nitrogen under weak alkaline to neutral conditions for germination, regardless of their different areas of occurrence. The former two species had a wider concentration of NH4Cl solution and pH range for germination in comparison to the latter two species. The optimum concentration of NH4Cl solution for the germination was 0.01 M in C. austrophlyctidospora and 0.1 M in the other three species. The pH optimum for germination in the former two species was 8.0 whereas that for germination in the latter two species was 8.0–8.5. The temperature range (5.0–40.0°C) for the former two species was wider than that (5–30°C) for the latter two species. Temperature optima for the germination in the former two species, C. aff. rugosobispora and C. echinospora, were 30, 20–25 and 15°C, respectively. The germination abilities of these Coprinopsis species in a wide range of temperatures are relevant to their natural temperature regime, showing their potential ability to propagate in tropical to subarctic regions.

Basidiospore germination by repetition and saprotrophic mycelia in the gelatinous telial horns of Gymnosporangium sabinae

AbstractThe teliospores of Gymnosporangium sabinae facultatively germinate with basidia or with haploid hyphae. The germination of basidiospores is by means of secondary spores (by repetition) or with haploid hyphae. In the gelatinous telial horns a saprotrophic mycelium is developed, that infected probably the host of the dikaryophase. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Effects of environmental factors on basidiospore germination of ammonia fungi <i>Coprinopsis</i> spp. collected from different geographical areas

Effects of pH, NH4-N, and temperature on basidiospore germination in Coprinopsis austrophlyctidospora from New Zealand, C. phlyctidospora from Japan, C. aff. rugosobispora from Canada, and C. echinospora from Canada were investigated. The Coprinopsis spp. required the presence of ammonium-nitrogen under weak alkaline to neutral conditions for germination, regardless of their different areas of occurrence. The former two species had a wider concentration of NH4Cl solution and pH range for germination in comparison to the latter two species. The optimum concentration of NH4Cl solution for the germination was 0.01 M in C. austrophlyctidospora and 0.1 M in the other three species. The pH optimum for germination in the former two species was 8.0 whereas that for germination in the latter two species was 8.0–8.5. The temperature range (5.0–40.0°C) for the former two species was wider than that (5–30°C) for the latter two species. Temperature optima for the germination in the former two species, C. aff. rugosobispora and C. echinospora, were 30, 20–25 and 15°C, respectively. The germination abilities of these Coprinopsis species in a wide range of temperatures are relevant to their natural temperature regime, showing their potential ability to propagate in tropical to subarctic regions.

Isolation and In Vitro Cultivation of Auriculoscypha anacardiicola D.A. Reid et Manim., an Insect-Associated and Potentially Medicinal Fungus from India

Auriculoscypha anacardiicola, an obligate insect-associate and a potential medicinal fungus, is isolated and studied in vitro. Suitable methods for isolation and cultivation of the fungus have been developed. Incubating spore deposits made to fall from basidiomata on tap water agar seems to be the best method for developing cultures. Successful isolations were also accomplished from infected coccids. Cultures could not be developed from single basidiospores and from tissues of the basidiomata. Although production of ballistospores and blastospores as well as germ tube formation were observed at the time of germination of basidiospores, budding blastospores alone produced mycelial cultures. Observations such as the inability of single basidiospores to germinate, emergence of mycelium from a spore deposit, and the apparent conjugation of yeast cells indicate that dikaryotization resulting from fusion of compatible yeast cells is essential for development of mycelium in A. anacardiicola. The fungus grew well on all complex media tested. It seems that a purely synthetic medium devoid of any growth factors cannot support the growth ofA. anacardiicola and yeast extract seems to provide the required growth factors.

Ecology of Hericium cirrhatum, H. coralloides and H. erinaceus in the UK

Hericium cirrhatum, Hericium coralloides and Hericium erinaceus are rarely recorded in Europe, and H. coralloides and H. erinaceus are Red Listed in several European countries. By contrast, H. erinaceus is common in nature in Japan and North America and easily cultivated for edible mushroom production. The three species are saprotrophs that grow on wood of broadleaved trees, particularly beech (Fagus sylvatica) in Britain. How they establish in the standing tree is uncertain, but they have been detected as latently present in functional sapwood. They frequently fruit in axenic culture, perhaps indicating that lack of fruit bodies in the field truly indicates lack of mycelial individuals, although it may be that natural fruiting is impeded by encapsulation within central woody tissues. Despite prolific sporulation in wild fruit bodies, basidiospore germination on agar is poor and large progeny sets of homokaryons were relatively difficult to obtain. The duration of the homokaryotic phase is unknown but may be prolonged due to low frequency of contact between mating compatible partners in natural substrata. Homokaryotic mycelia grow at least as rapidly as heterokaryons. Both homokaryons and heterokaryons are average to good combatants, the former slightly better than the latter. Poor growth and combative ability are unlikely to be the causes of apparent rarity in Europe, but poor germination and infrequent establishment may be.

A Tetrad Analysis of the Basidiomycete Fungus Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete fungus that is found worldwide and causes disease in humans and animal species. The fungus grows asexually as a budding yeast. Under laboratory conditions it is capable of sexual reproduction between two mating types. After cell fusion a dikaryotic filament develops, at the tip of which a basidium gives rise to four chains of basidiospores. Because the chains each comprise 10-30 spores, rather than single spores, the analysis of individual meiotic events has not been attempted in C. neoformans in the style of tetrad analyses performed in other fungal species. Here, the basidiospores from >100 basidia were micromanipulated and the resultant >2500 progeny analyzed for three genetic markers to understand the sexual process in this fungus, leading to four observations: (i) Marker segregation provides genetic evidence for a single meiotic event within the basidium followed by multiple rounds of mitosis. (ii) Using each basidium as an unordered tetrad, the ADE2 and URA5 genes are linked to their centromeres, consistent with adjacent genomic regions rich in repetitive elements predicted to comprise Cryptococcus centromeres. (iii) Lack of germination of basidiospores is attributed to aneuploidy, rather than dormancy. (iv) Analysis of basidiospores derived from single chains demonstrates that each chain can contain different genotypes. This mechanism of sexual spore production would benefit the species with a high rate of dispersal and at the same time aid in simultaneous dissemination of both mating types to new locations in the environment.

The rare oak polypore Piptoporus quercinus: Population structure, spore germination and growth

Piptoporus quercinus, the rare oak polypore, is one of only four non-lichenised fungi listed on Schedule 8 of the Wildlife and Countryside Act 1981, in the UK. It is thereby accorded the highest level of legal protection in the UK. It fruits exclusively on oak (Quercus spp.), primarily veteran trees in old growth wood pasture and parkland in the midlands and southern England. Beyond information on fruit body occurrence, little is known of the ecology of this species. Germination of chlamydospores and basidiospores was investigated under a range of conditions (temperature, pH, different media). The mating system was ascertained by pairing single spore derived isolates, and somatic compatibility investigated by pairing fruit body isolates. Germination of basidiospores was consistently below 1 % with most success occurring on 2 % malt extract agar adjusted to pH 4 at 20 °C. Chlamydospores had a much higher average percentage of germination (47 % ± 22), and germinated even after prolonged exposure to extreme conditions (desiccation or exposure to −20 °C or 40 °C for 48 h, 7 d or 14 d). The mating system was unifactorial (bipolar), although only four mating alleles were discovered in the six populations sampled, two alleles being unique to one fruit body. There were no signs of somatic incompatibility between the majority of isolates, exceptions being an isolate from Wentwood, South Wales, and an isolate from Germany collected in 1975, both of which displayed apparent signs of somatic incompatibility in all experimental pairings. The lack of somatic incompatibility between the majority of UK isolates, together with the low number of mating alleles, may point to inbreeding within the UK population. Apparently compatible isolates were not, however, clonal, as shown by variation in their mycelial extension rates on agar at various temperatures.

Production of lytic enzymes and siderophores, and inhibition of germination of basidiospores of Moniliophthora (ex Crinipellis)perniciosa by phylloplane actinomycetes

Streptomyces albovinaceus, Streptomyces caviscabies, Streptomyces griseus, Streptomyces setonii, and Streptomyces virginiae selected as antagonists of Moniliophthora (ex Crinipellis) perniciosa, the causal agent of cacao Witches’ broom, were examined in vitro to detect production of chitinases, β-1,3-glucanases, and cellulases. All the species produced chitinases, but not β-1,3-glucanases or cellulases, when grown on a liquid mineral medium containing glucose, colloidal chitin, or cell walls of M. perniciosa as a carbon source. There were no quantitative differences among species in the production of chitinase, however, the germination inhibition of basidiospores of M. perniciosa was higher when they were cultivated using glucose as a carbon source, followed by colloidal chitin and cell walls. All the species also produced hydroxymate type siderophores in similar quantities, and the quantity of siderophores did not correlate with the inhibition of basidiospore germination. The germination inhibition was more pronounced when S. albovinaceus, S. griseus, and S. virginiae were cultivated on iron-deficient medium, suggesting involvement of siderophores in the antagonism by these species of actinomycetes.

Effects of pH, NH4-N concentration, temperature, and storage period on basidiospore germination in an ectomycorrhizal ammonia fungus Hebeloma vinosophyllum

Basidiospore germination in an ectomycorrhizal ammonia fungus Hebeloma vinosophyllum was stimulated by 10–500mM NH4Cl aqueous solution at pH 4.5–9.0, but not by pure water. The basidiospores germinated at 10°–35°C with an optimum at 25°–30°C. The highest germination percentage (83.0%) was observed in 100mM NH4Cl aqueous solution adjusted to pH 8.0 by KOH, when the basidiospores were incubated at a density of 106 spores/ml at 30°C for 14 days. The percent germination value decreased with the increased duration of storage under both dry and wet conditions. Humidity and temperature affected the longevity of H. vinosophyllum basidiospores. The basidiospores maintained their germination ability longer under a dry condition than under a wet condition. The greatest longevity was accomplished by storage at 15°C under a dry condition.

Bipolar incompatibility system of an ectomycorrhizal basidiomycete, Rhizopogon rubescens

The mating systems of most ectomycorrhizal fungi have not been elucidated because of two reasons. One is the difficulty of obtaining homokaryotic isolates for mating tests caused by the low germination rate of basidiospores, and another is the difficulty of checking dikaryotization caused by the absence or inconsistent production of clamp connections on heterokaryotic mycelia under laboratory conditions. Basidiospore germination of a few ectomycorrhizal fungi has been induced by living roots of their host plants. Based on this information, we examined methods to obtain homokaryotic isolates of Rhizopogon rubescens using its host plant, Pinus thunbergii. The basidiospores of R. rubescens appeared to germinate well on an agar plate, on which axenic pine seedlings were grown in advance to induce germination, even when the seedlings were removed from the plate at the time of spore inoculation. To enhance the production rate of clamp connections on the heterokaryotic mycelia of R. rubescens, the culture medium composition was modified. The pH of the medium was critical for the production of clamp connections, and the optimal pH was higher for the production of clamp connections than for mycelial growth. These findings made it possible to conduct mating tests, and we found that the mating system of R. rubescens is bipolar with a multiallelic mating type factor.