An Analysis of Spore Discharge in Sordaria

Sordaria fimicola can develop mature perithecia from which sporesare discharged when grown in darkness or in light. Under conditionsof alternating dark and light (12 hrs.: 12 hrs.) each day, sporedischarge is periodic with a low rate during the dark period,succeeded by a gradual rise to a relatively high rate in thelight period followed by a decline before the onset of the nextdark period. There is no trace of an endogenous rhythm. Transferfrom darkness to light always leads to an increase in the rateof discharge, and from light to dark to a decrease. The heightof the peak of discharge rate attained in light following adark period seems to be related to the length of the precedingdark period. Experiments with light of different colours but of roughly thesame energy value show that it is the blue rays that are mainlyeffective. From cultures of filter-paper yeast-extract mediuman orange pigment can be extracted with a maximum absorption,in the visible spectrum, at 470 mµ. It is possible thatthis is important in connexion with the sensitivity of the fungusto blue light.     

Stimulation of Spore Discharge by Reduced Humidity in Sordaria

An account is given of experiments on the effect of the humidityof an ambient air-stream on spore discharge in Sordaria fimicola.Change from saturated to dry (35 per cent. R. H.) conditionsinvariably led to a striking increase in the rate of spore discharge,and conversely change from dry to damp conditions produced amarked decrease.     

An Endogenous Rhythm of Spore Discharge in Sordaria fimicola

The sporulation process in Sordaria fimicola has been studiedunder controlled conditions, with particular reference to discharge.Discharge occurred with a circadian periodicity for the greaterpart of the sporulation life of the culture, with peaks at about18.00 hours in either light or darkness. The consistency ofthis phenomenon was confirmed by an analysis of previous work.Fluctuations in light intensity, temperature, relative humidity,carbon dioxide concentration, and vibration were eliminatedas far as possible, as causes of this periodicity. Minor fluctuationsin discharge rate with a period of less than 24 h were probablyrandom. The general nature of the periodicity was shown to be consistentwith some aspects of endogenous rhythms in other organisms.However, spore discharge in S. fimicola is very sensitive tolight, and it is readily entrained to discharge out of phasewith the natural period. The rate of development of two stages of spore formation (delimitationand maturation) were determined, and these were related to therate and periodicity of discharge. From the data it was impossibleto establish at which stage of sporulation the periodicity arose.     

Stimulation of Spore Discharge in Sordaria by Carbon Dioxide

The effect on the rate of spore discharge in Sordaria fimicolaof small concentrations of carbon dioxide has been studied.Substitution of an air-stream containing 0.2-2.5 per cent carbondioxide for air freed from this gas circulating over the peritheciaalways led to a marked increase in the rate of spore discharge.     

Effects of Airspeed and Humidity Changes on Spore Discharge in Sordaria fimicola

An improved experimental system is described for studying therate of spore discharge in Sordaria fimicola subjected to anair-stream of controlled rate and relative humidity. Under nearlysaturated conditions the rate of air-flow does not seem to affectthe rate of discharge. Reduction of the R.H. from 95 per centto 70 per cent or 35 per cent leads to an immediate but temporaryincrease in discharge rate. 70 per cent R.H. seems only to affectactual discharge, but prolonged exposure to 35 per cent R.H.interferes as well with earlier stages in spore development.It is suggested that the immediate effect on discharge is physicalrather than biochemical. There is evidence that the perithecialwall plays some part in the control of spore discharge.     

Light and Spore Discharge in Ascobolus

Spore discharge in Ascobolus crenulatus occurs both in the lightand in the dark. In a 12 h light: 12 h dark daily regime discharge-ratehas peaks in the dark periods, due apparently to light stimulationwith about half a day's interval between stimulus and response. Using a ‘spore clock’ the course of discharge hasbeen followed for a single apothecium on changing from darknessto light. Exposure to light (500 lux) of wave-lengths around400, 440 and 460 mµ immediately causes ’puffing‘,whilst light of longer wave-length (504 and 580 mµ) hasno effect. Change from darkness to white light has no immediateeffect, but there is a delayed stimulation with a marked increasein discharge-rate 10–14 h later. Simultaneous illuminationof an apothecium, which has been in darkness, by blue light(420 mµ, or 440 mµ, 500lux) and yellow light (580mµ, 500 lux) does not result in puffing. The yellow appearsto prevent the blue light from exerting its effects.     

Periodicity of Spore Discharge in Daldinia

In Daldinia concentrica spore discharge under natural conditionsis periodic, most spores being discharged during the night andfew in the daytime. An experimental study has been made of dischargeunder controlled conditions of light and temperature. In continuousdarkness periodic discharge was maintained for 12 days, butthen, although spore output continued, it ceased to be periodic.Return to alternating light (12 hrs., 100 f.c.) and darkness(12 hrs.) at once re-established the periodicity. In continuouslight (100 f.c.) periodic discharge ceased after 2 to 3 days,but was immediately re-established in alternating light (12hrs.) and darkness (12 hrs.). When the fungus was placed underconditions of alternating light and darkness each of 6 hours'duration, two peaks of spore-output were soon developed in the24-hour period. The experiments suggest that the natural periodicityis determined by the alternation of day and night.     

Spore Discharge in the Basidiomycetes: I. Periodicity of Spore Discharge by Trametes gibbosa Fr.

Variations in the rates of spore discharge from this fungushave been examined in controlled laboratory conditions, usingapparatus designed to make a continuous record of the numbersof spores discharged by a strip of hymenium measuring approximately4.5 cm. X o.1 cm. These records have been extended by a directmicroscopic study of the numbers of spores falling through asmall area below the hymenium during successive short periods. Analysis of the continuous record has shown that at any onetime the rate of spore discharge is not uniform over the wholehymenium, and that at any one site it ia not constant. The variationin the rate of discharge in any one site is rhythmical, withintervals of the order of 30 to 90 minutes between maxima. Thesmall linear areas of hymenium which have been examined haveshown nearly the same rhythm-overall, but the phase of thisrhythm has differed at different sites in the area.     

Spore Discharge in Basidiomycetes: III. Spore Liberation from Narrow Hymenial Tubes

The hymenial tubes of Polyporus betulinus were shown to be approximatelycylindrical,and to have a sporulating hymenium over the greater part oftheir surface. Sporophores were tilted through measured anglesfrom the vertical in the field and in the laboratory, and theeffect on spore liberation was measured by determining the numberof spores liberated from particular tubes in standard time.All tilting caused a reduction in spore liberation. The amountof this reduction was found to compare closely with that predictedfrom calculation of the amount of hymenium lying directly abovethe orifice at various angles of tilt. It is therefore concludedthat liberation from the tube can be accounted for by the initialviolent discharge of the spore and gravitational attractionalone. An addendum corrects an earlier paper (1959) in thisseries. It shows that the variation in density of spore depositscollected from Trametes gibbosa was due to air currents in theapparatus, not to variation in sporulation rate.     

Spore Discharge in Deightoniella torulosa (Syd.) Ellis

Violent spore discharge in the Hyphomycete Deightoniella torulosais described. On drying, a negative pressure develops in thesolution inside the swollen head of the conidiophore. This causesthe thin wall at the top of the conidiophore to cave inwardsand to be brought into a state of tension. The sudden appearanceof a gas phase in the conidiophore head releases this tension,and the wall rapidly returns to its original rounded shape.The conidium, which is attached to the top of the conidiophore,is thus catapulted away. This appears to be the first recordof a gas phase being associated with violent spore dischargein a fungus.     

Adaptation of the Spore Discharge Mechanism in the Basidiomycota

Background

Spore discharge in the majority of the 30,000 described species of Basidiomycota is powered by the rapid motion of a fluid droplet, called Buller''s drop, over the spore surface. In basidiomycete yeasts, and phytopathogenic rusts and smuts, spores are discharged directly into the airflow around the fungal colony. Maximum discharge distances of 1–2 mm have been reported for these fungi. In mushroom-forming species, however, spores are propelled over much shorter ranges. In gilled mushrooms, for example, discharge distances of <0.1 mm ensure that spores do not collide with opposing gill surfaces. The way in which the range of the mechanism is controlled has not been studied previously.

Methodology/Principal Findings

In this study, we report high-speed video analysis of spore discharge in selected basidiomycetes ranging from yeasts to wood-decay fungi with poroid fruiting bodies. Analysis of these video data and mathematical modeling show that discharge distance is determined by both spore size and the size of the Buller''s drop. Furthermore, because the size of Buller''s drop is controlled by spore shape, these experiments suggest that seemingly minor changes in spore morphology exert major effects upon discharge distance.

Conclusions/Significance

This biomechanical analysis of spore discharge mechanisms in mushroom-forming fungi and their relatives is the first of its kind and provides a novel view of the incredible variety of spore morphology that has been catalogued by traditional taxonomists for more than 200 years. Rather than representing non-selected variations in micromorphology, the new experiments show that changes in spore architecture have adaptive significance because they control the distance that the spores are shot through air. For this reason, evolutionary modifications to fruiting body architecture, including changes in gill separation and tube diameter in mushrooms, must be tightly linked to alterations in spore morphology.     

Further Observations on Light and Spore Discharge in Certain Pyrenomycetes

A ‘spore-clock’ for studying the hourly rate ofspore discharge over a 24-hour period is described. A numberof the experiments reported in this paper have involved theuse of this apparatus. In Sordaria fimicola there is a distinct positive light-dischargereaction in a dark-conditioned culture, the rate of spore dischargeincreasing steeply to a peak 2–3 hours after brief stimulationby bright light. Although darkening a light-conditioned cultureleads to an immediate decrease in the rate of discharge, thereis no evidence of a delayed negative dark-discharge reaction. In S. verruculosa with a 12-hours light: 12-hours dark dailyreëgime, more spores are discharged in the dark than inthe light periods if the intensity of illumination is low. Withhigher light intensity there is no significant difference betweenthe number of spores discharged in light and dark periods. Asin S. fimicola there is a positive light-discharge reaction,the interval between stimulus and maximum response being muchlonger (8–12 hours). When a dark-conditioned culture istransferred to light for 48 hours and then returned to darknessfor a further 48 hours it is apparent that not only is therea positive light-discharge reaction but also a negative dark-dischargeresponse. The ‘plateau’ level of discharge is essentiallythe same in light and darkness. It is confirmed that in Hypoxylon fuscum light inhibits discharge.     

Effect of Light on Spore Discharge of Selected Basidiomycetes

Following entrainment of nine selected species of basidiomyceteswith alternating 12 h light and 12 h dark periods, circadianrhythms with nocturnal peaks of spore discharge were found topersist in continuous light or dark for periods of 4 –9d. However, when light and dark periods were reversed followingentrainment, Piptoporus belulinus. Panellus stipticus, Awiculariaauricula and Dacrymyces deliquescens made immediate readjustmentof their rhythms; Coriolus Dersicolor, Stereum hirsutum andClitocybe nebularis took 24 h to readjust with peaks of sporedischarge synchronized with the new dark time periods, whileGanoderma applanatum and Trametes betulina took 48 h to readjust.These reactions indicate exogenous rhythms Light effects, spore discharge, basidiomycetes     

Perithecium development in Sordaria brevicollis

Microconidia and ascogonial coils were produced by the two strains of Sordaria brevicollis , WTA and WTa. Over 90% of the microconidia, which functioned chiefly for sexual reproduction, germinated producing short–lived germ tubes. Ascogonial coils with conspicuous trichogynes were observed. A hypha was initiated from the base of the ascogonial coil and soon completely surrounded it, giving rise to the protoperithecium. The ascogonial coil became the ascogonium within the proto–perithecium, and it was surrounded by a pseudoparenchymatous centrum. Many paraphyses arose from pseudoparenchymatous cells. The ascogonium followed the Sordariaceous type of development and gave rise to ascogenous hyphae, croziers, unitunicate asci and ascospores. Anomalous perithecia were observed and perithecia reached maturity 9–1 1 days after inoculation.     

Perithecium morphogenesis in Sordaria macrospora

The perithecium of the self-fertile ascomycete Sordaria macrospora provides an excellent model in which to analyse fungal multicellular development. This study provides a detailed analysis of perithecium morphogenesis in the wild type and eight developmental mutants of S. macrospora, using a range of correlative microscopical techniques. Fundamentally, perithecia and other complex multicellular structures produced by fungi arise by hyphal aggregation and adhesion, and these processes are followed by specialization and septation of hyphal compartments within the aggregates. Perithecial morphogenesis can be divided into the ascogonial, protoperithecial, and perithecial stages of development. At least 13 specialized, morphologically distinct cell-types are involved in perithecium morphogenesis, and these fall into three basic classes: hyphae, conglutinate cells and spores. Conglutinate cells arise from hyphal adhesion and certain perithecial hyphae develop from conglutinate cells. Various hypha-conglutinate cell transitions play important roles during the development of the perithecial wall and neck.     

Segregation of genes in Sordaria brevicollis

The segregation of five U.V. induced morphological marker genes and mating-type has been studied in Sordaria brevicollis. Two peculiar morphological markers crisp (cr) and dic (dichotomous terminal mycelial branching) were assigned to linkage groups I and II respectively.The results show that partial spindle overlap which is thought to be the cause of the occurrence of a significant excess of asymmetrical post-reduction tetrads over symmetrical types might not be interfering significantly with the pattern of segregation of genes in this organism. It is suggested that normal crossing-over events could account for the occurrence of a significant excess of asymmetrical post-reduction tetrads for proximal marker genes.     

Microsporidian Spore Discharge and the Transfer of Polaroplast Organelle Membrane into Plasma Membrane

Electron microscopic examinations of Glugea hertwigi and Spraguea lophii spores indicated the presence of a single plasma membrane; however, this membrane remained in the spore during the discharge of the sporoplasm from the spore. Although discharged spores retained the old plasma membrane, the extruded sporoplasms acquired a new plasma membrane. In order to determine where the new plasma membrane came from, we used two fluorescent probes with membrane affinities. The markers were tested on unfired and discharged spores. The probe, N-phenyl-1-naphthylamine (NPN), labeled the polaroplast membrane in addition to the apolar groups in the posterior vacuoles of unfired spores. After spore discharge, NPN label disappeared from the spore ghosts except for a slight fluorescence on residual plasma membranes. Much of the NPN-labeled membrane reappeared after spore discharge on the outer envelope of discharged sporoplasms. The probe chlorotetracycline (CTC) labeled calcium-associated membranes of spore polaroplasts. During spore discharge, the CTC fluorescence shifted from the polaroplast organelle of unfired spores to the outer envelope of discharged sporoplasms. These results indicate that the polaroplast organelle may provide the new plasma membrane for discharged microsporidian sporoplasms.