Ascospore Release Research Articles

Phenology of development of Didymella rabiei pseudothecia and modelling of ascospores discharge in Tunisia

Didymella rabiei development was investigated on naturally blight-infested debris in six Tunisian locations during two consecutive seasons. Pseudothecia initiated their development 2 months after their incubation on the soil surface. They reached their maturity earlier in the locations of Korba, Beja, Bizerte and Bousalem (March–April) than in Morneg and Tunis (June). After ascospore discharge, new asci and ascospores did not develop in empty pseudothecia, and the fungus survived during the second season by forming pycnidia. Pseudothecia maturity index (PMI) was determined based on internal developmental stages and a logistic model was proposed to establish an eventual correlation between PMI and Celsius degree days (CDD) cumulated over time during rainy days (for rain ≥1 mm). Results showed that PMI increased significantly over cumulative number of CDD in the six locations. In Korba, Beja, Bizerte and Bousalem locations, maximum rate of pseudothecia maturity occurred during an acceleration phase between 150 and 350 cumulated CDD during which pseudothecia reached the mature stage. The maturity rate of pseudothecia in Morneg and Tunis locations was slower as compared to the latter four locations and mature pseudothecia were observed at 450–500 cumulated CDD. The onset of pseudothecial maturity was also estimated by applying a pre-established model previously developed for Ascochyta disease management. Based on this model, which considers a combination of daily mean temperature and daily total rainfall, the number of suitable events required for pseudothecial maturity was determined at each location.

Forecasting system for infection risk of phoma stem canker in selected regions of the Czech Republic in 2009-2011

In the period 2009–2011 monitoring of the incidence of phoma stem canker was carried out at selected sites of the Czech Republic (Šumperk and Opava regions in North Moravia). The risk of infection was evaluated by assessing the autumn release of Leptosphaeria spp. ascospores and with the proPlant prediction model. In recent years, the incidence of phoma stem canker has been relatively low and this corresponded with the total count of ascospores released in the autumn period but not with an increased level of infection risk announced by the proPlant model. During the monitored period the ascospore concentration reached maximally 2 ascospores/1 m<sup>3</sup> per day. Maximum ascospore release was recorded in 2010 as a result of favourable weather conditions. The first incidence of phoma leaf spot has been observed in mid-October. The higher values of ascospores concentration were recorded in the Opava region, but the total number of the trapped ascospores was more often higher in the Šumperk region. 

A Quality Control Mechanism Linking Meiotic Success to Release of Ascospores

Eukaryotic organisms employ a variety of mechanisms during meiosis to assess and ensure the quality of their gametes. Defects or delays in successful meiotic recombination activate conserved mechanisms to delay the meiotic divisions, but many multicellular eukaryotes also induce cell death programs to eliminate gametes deemed to have failed during meiosis. It is generally thought that yeasts lack such mechanisms. Here, we show that in the fission yeast Schizosaccharomyces pombe, defects in meiotic recombination lead to the activation of a checkpoint that is linked to ascus wall endolysis – the process by which spores are released in response to nutritional cues for subsequent germination. Defects in meiotic recombination are sensed as unrepaired DNA damage through the canonical ATM and ATR DNA damage response kinases, and this information is communicated to the machinery that stimulates ascus wall breakdown. Viability of spores that undergo endolysis spontaneously is significantly higher than that seen upon chemical endolysis, demonstrating that this checkpoint contributes to a selective mechanism for the germination of high quality progeny. These results provide the first evidence for the existence of a checkpoint linking germination to meiosis and suggest that analysis solely based on artificial, enzymatic endolysis bypasses an important quality control mechanism in this organism and potentially other ascomycota, which are models widely used to study meiosis.

Neurospora crassa ncs-1, mid-1 and nca-2 double-mutant phenotypes suggest diverse interaction among three Ca2 + -regulating gene products

The calcium (Ca2+) signalling system in the filamentous fungus Neurospora crassa is unique and significantly different from that in plants and animals, mainly with regard to the second messenger systems involved in Ca2+-release from internal stores (Galagan et al. 2003). The complex Ca2+ signalling system in N. crassa contains 48 Ca2+ signalling proteins including a Ca2+ and/or CaM binding protein called neuronal calcium sensor-1 (NCS-1), a Ca2+-permeable channel MID-1 and a PMCA-type Ca2+-ATPase NCA-2 (Zelter et al. 2004; Tamuli et al. 2013). Here, we show that ncs-1, mid-1 and nca-2 interactions affect growth, carotenoid accumulation, Ca2+ stress tolerance, ultraviolet (UV) survival and circadian-regulated conidiation in N. crassa. The N. crassa homologue of NCS-1 has a role in growth, Ca2+ stress tolerance and UV survival (Deka et al. 2011; Tamuli et al. 2011). MID-1 is necessary for Ca2+homeostasis in N. crassa (Lew et al. 2008). Mid-1 in Gibberella zeae has a role in growth, development and ascospore discharge (Cavinder et al. 2011). NCA-2 is responsible for adaptation to stress conditions and functions to pump Ca2+ out of the cell in N. crassa (Benito et al. 2000; Bowman et al. 2011). In Schizosaccharomyces pombe, Ncs1p, the homologue of NCS-1, promotes Ca2+-induced closure of Yam8p Ca2+ channel, a homologue of the MID1 (Hamasaki-Katagiri and Ames 2010). The Ca2+-sensitive phenotype of the ncs1 deletion mutant is rescued by a yam8 deletion, indicating that Ncs1p negatively regulates Yam8p in S. pombe

Role of ascospores and conidia in the initiation and spread of South American leaf blight in a rubber tree plantation

Microcyclus ulei, the fungus causing South American leaf blight (SALB) on rubber tree leaves, produces two main types of spores: ascospores and conidia. To assess their respective epidemiological role, a field experiment was conducted in French Guiana over 3 years. Tree phenology, disease severity and climate variables were recorded while airborne spores were trapped and quantified. Ascospores were shown to play an essential role in the perpetuation of the disease outside the host's growth periods, in the resumption of epidemics, and in the spread of the disease to disease‐free zones. Conidia were trapped in visibly infected plots only, during periods of host growth. Disseminated over short distances and present only temporarily on leaves, the conidia enabled the disease to spread stepwise when the climate was conducive. Segmentation analysis revealed that the duration of high relative humidity was the climatic variable most related to ascospore trapping. Ascospore release did not require low temperatures. Considering the essential role of the ascospores in the initiation and spread of disease, artificial defoliation as a means of reducing the inoculum pressure during tree refoliation is proposed to control SALB. To check the validity of this method, a survey over several years of natural defoliation–refoliation in relation to climate and other leaf diseases is needed.

Predicting the risk of cedar leaf blight (Didymascella thujina) in British Columbia under future climate change

Cedar leaf blight (Didymascella thujina) is considered to be the most important disease of western redcedar in British Columbia. The disease is most prevalent in warm-moist coastal low-elevation environments causing mortality among seedlings and significant loss of incremental growth and branch death among mature trees. In this study we used a principle component regression model to spatially project the disease risk under observed climate (2003–2008) and multiple future climate scenarios for the 2020s, 2050s, and 2080s. We found that while the majority of future climate scenarios predicted coastal environments will continue to favor occurrence of the disease, intensity is predicted to decrease toward the 2080s. Projected reductions of available summer climate moisture (cumulative precipitation – potential evapotranspiration), corresponding to the time of ascospore discharge and germination, contribute significantly to this finding. Disease intensity is however, projected to increase under moderate temperature and precipitation increases for the 2020s. We therefore recommend current reforestation efforts deploy disease resistant western redcedar seedlots in high risk environments common to hypermaritime coastal regions such as Haida Gwaii and northern maritime, to avoid significant mortality and growth reduction.

The invasive ash dieback pathogen Hymenoscyphus pseudoalbidus exerts maximal infection pressure prior to the onset of host leaf senescence

Shoot dieback disease of European ash caused by the ascomycete Hymenoscyphus pseudoalbidus threatens ash on a continental scale. A spore sampler placed in a diseased ash forest in Southern Norway, coupled with microscopy and DNA-based fungal species-specific real-time PCR assays, was employed to profile diurnal and within-season variation in infection pressure by ascospores of H. pseudoalbidus and the potentially co-existing non-pathogenic Hymenoscyphus albidus. Hymenoscyphus pseudoalbidus was found to be predominant in the stand. Massive simultaneous liberation, by active discharge of pathogen ascospores in the morning, peaked in mid-Jul. to mid-Aug. Accumulation of pathogen DNA on leaflets of current-year leaves reached a high level plateau phase before appearance of autumn coloration, suggesting that pathogen establishment in leaves is terminated before the onset of leaf senescence.

The “firing cannons” of Dipodascopsis uninucleata var. uninucleata

According to literature, the elongated ascospores of Dipodascopsis uninucleata var. uninucleata exhibit smart movement when forcefully ejected from bottle-shaped asci. This type of movement is defined as the unique patterns of non-random movement of ascospores with specialized morphology thereby facilitating release from asci. Smart movement is required to actively release ascospores individually through the narrow ascus neck, without causing an obstruction and blocking ascospore release. However, little is known about the propulsion mechanism of this cannon-type release system. We show that asci of this yeast contain a central channel (barrel) filled with ascospores. These are surrounded by a sheath-like structure that lines the inner surface of the ascus wall. We found that this sheath is responsible for forcing the naked ascospores out of the ascus by exerting turgor pressure from the bottom towards the tip of the ascus. This cannon firing system is in contrast to that found in Dipodascus geniculatus, where no sheaths lining the ascus interior were observed. Instead, sheaths were found enveloping each ascospore.

Overview of some recent research developments in fusarium head blight of wheat

This article reviews the recent progress of research on fusarium head blight (FHB) of wheat. It addresses the broad areas of strategies for disease management, biological control, the pathogen (Fusarium graminearum (teleomorph Gibberella zeae)), mycotoxins, the effects of dwarfing genes on FHB severity, quantitative trait loci (QTLs) and new perspectives. Where there are recent reviews on this subject, we have deliberately examined the subsequent literature to provide an update on research. With few resistant cultivars available even now, the main tools to manage the disease remain rotation, varietal selection, disease forecasting and fungicides. A few biocontrol organisms are being considered for commercial application. The pathogen's sexual life cycle has been investigated in depth, and with its complete genome sequence known, the pathways and genes controlling the sexual development and ascospore release of F. graminearum are being explored. The 3-acetyldeoxynivalenol chemotype of F. graminearum has increased in prevalence in Canada with attendant risks of higher DON levels in cereal grain. Stringent limits on allowable levels of Fusarium mycotoxins in the food/feed chain have been enacted in Europe and the USA, but regulations for Canada are only at the discussion stage with the Canadian Food Inspection Agency. Efforts to develop FHB-resistant lines proceed apace, as these can be selected in most wheat populations despite the adverse effects of dwarfing genes on FHB severity. While more quantitative trait loci (QTLs) for disease resistance continue to be identified and mapped, new resistant cultivars remain disappointingly few. We present some encouraging early results from an alternative approach based on epigenetics.

Modeling the Effect of Temperature and Wetness on Guignardia Pseudothecium Maturation and Ascospore Release in Citrus Orchards

Ascospores are the most important inoculum source of citrus black spot (CBS), caused by Guignardia citricarpa, but pseudothecium maturation and ascospore release are inadequately studied. Guignardia ascospore trapping and concomitant weather data were obtained for three localities over three seasons (July to March 2006 to 2009) in the Limpopo province of South Africa. Degree-days accumulated until first seasonal ascospore discharge (>10°C with 1 July as biofix) (DDtemp), and DDtemp accumulated on rainy (rainfall >0.1 mm) (DDrain) and moist days (vapor pressure deficit <5 hPa) (DDvpd) were used in two Gompertz models to predict onset of ascospore release: a temperature model [Event = exp(-exp(-(-2.725 + 0.004 × DDtemp)))] and a temperature/moisture model [Event = exp(-exp(- (-3.238 + 0.008 × DDvpd + 0.004 × DDtemp - 0.009 × DDrain)))] (R(2) = 0.608 and 0.658, respectively). Both models predicted a delay in pseudothecium maturation in climates with colder winters and springs. A Gompertz equation was also used to predict the proportion of Guignardia ascospores trapped (PAT) per season from DDtemp data accumulated on wet or moist days (DDwet2) from the first seasonal ascospore discharge [PAT = exp(-4.096 × exp(-0.005 × DDwet2); R(2) = 0.908]. The PAT model predicted lag phases and 7-day peaks in ascospore release patterns with reasonable accuracy. These models can be used to predict the onset and dynamics of ascospore release in climatically diverse regions.

Evaluation of three models for predicting &lt;i&gt;Venturia inaequalis&lt;/i&gt; ascospore release in Southern Brazil

Ascospores of Venturia inaequalis constitute the principal source of primary inoculum for epidemics of apple black spot (scab) Mathematical models that describe ascospore release have been developed in USA New Zealand and Italy These models are based upon degreeday accumulation In this work the seasonal pattern of released ascospores of V inaequalis in Vacaria Brazil was evaluated During four apple growth seasons (2009 to 2012) the natural release of ascospores from five orchards was recorded The cumulative percentage of ascospore release was predicted using three different degreeday models The released ascospore data showed the best fit to the model developed in Italy with a start date of 22 August rather than the phenological stage of apple bud break It is concluded that in regions with warm winter temperatures models that use bud break to initiate ascospore maturation will tend to underestimate availability of ascospores at the beginning of the apple growth season

&lt;i&gt;Leptosphaeria maculans/L biglobosa&lt;/i&gt; disease progression in oilseed rape and timing of ascospore release under New Zealand conditions

Leptosphaeria maculans/L biglobosa disease progression in oilseed rape and the timing of ascospore release from crop debris were studied in field experiments in 2011 and 2012 A similar pattern of disease progression was observed in both years with the first leaf lesions characteristic of L maculans observed 115 weeks after sowing and stem lesions after 25 weeks Leaf lesions characteristic of L biglobosa were observed after 175 weeks Leptosphaeria maculans was isolated from nonsymptomatic petioles and stems after 165 and 22 weeks respectively indicating endophytic growth of the pathogen from leaf lesions Vaselinecoated slides did not trap ascospores in 2011 but in 2012 both Rotorod and Burkard spore samplers trapped ascospores from 29 May to 21 September Nested PCR with ITS and speciesspecific primers detected both L maculans and L biglobosa ascospores with L maculans being more frequently detected Peak ascospore numbers were detected in August and associated with periods of high rainfall

Some topics in tan spot epidemiology - characteristic and significance the sources of primary infection

The aim of this studies was characteristic of the sources of primary infection for tan spot. During the period 1998-1991 the development of ascomata and conidimata of &lt;i&gt;Pyrenophora tritici-repentis&lt;/i&gt; on naturally infected wheat and triticale straws was monitored. The process of pseudothecia formation was extending over the time. It started immediately after harvest and protothecia of the fungus was commonly occurred each year already in the fall. The first fruit bodies with maturated typically pigmented ascospores were observed in January 2001 and in February 1998 and 1999. The percentage of pseudothecia with maturate ascospores successively increased richening maximum in April when the massive liberation of spores from asci occurred. After the total ascospore release on surface of empty ascomata aroused conidiophores with conidia which may also act as inoculum source. Moreover this type of sporulation was observed directly on over wintered cereals stems especially after prolonged period of rains. The present studies indicated also on infected by &lt;i&gt;P.tritici-repentis&lt;/i&gt; wheat and triticale seeds as a source of inoculum for tan spot.

Temporal Patterns of Ascospore Release in Leptosphaeria maculans Vary Depending on Geographic Region and Time of Observation

Diurnal patterns of spore release have been observed in a number of fungal pathogens that undergo wind-assisted dispersal. The mechanisms that drive these patterns, while not well understood, are thought to relate to the ability of dispersing spores to survive their journey and infect new hosts. In this paper, we characterise the diurnal pattern of ascospore release by a Western Australian population of Leptosphaeria maculans. Although L. maculans has been previously shown to exhibit diurnal patterns of ascospore release, these patterns appear to vary from region to region. In order to characterise the pattern of release in the Mediterranean climate of Western Australia, we analysed historical data describing the bi-hourly count of airborne ascospores at Mt Barker, Western Australia. Results of this analysis showed diurnal patterns that differ from those previously observed in other countries, with ascospore release in our study most likely to occur in the afternoon. Furthermore, we found that the time of peak release can shift from month to month within any one season, and from year to year. In explaining the hourly pattern of spore release over an entire season, time since rainfall, time since last release, temperature, hour and month were all shown to be significant variables.

Combining sanitation and disease modelling for control of grapevine powdery mildew

Chasmothecia of Erysiphe necator form in one season, survive winter and discharge ascospores that cause primary infections and trigger powdery mildew epidemics in the next season. A strategy for powdery mildew control was developed based on (i) the reduction in overwintering chasmothecia and on (ii) spring fungicide applications to control ascosporic infections timed based on estimate risk (two to five sprays per season). Several fungicides, the hyperparasite Ampelomyces quisqualis, and a mineral oil product were first tested as separate applications in a greenhouse and in vineyards. In the greenhouse, A. quisqualis suppressed chasmothecia formation by 41 %; fungicides and mineral oil suppressed chasmothecia formation by 63 % and ascospore viability by 71 %. In vineyards, application of boscalid + kresoxim-methyl or meptyldinocap once after harvest, as well as application of A. quisqualis pre- and post-harvest, delayed disease onset and epidemic development in the following season by 1 to 3 weeks and lowered disease severity (up to the pea-sized berry stage) by 56 to 63 %. Risk-based applications of sulphur and of synthetic fungicides provided the same control as the grower spray program but required fewer applications (average reduction of 47 %). Sanitation strategies were then tested by combining products and application times (late-season, and/or pre-bud break, and/or spring). Adequate disease control with a reduced number of sprays was achieved with the following combination: two applications of A. quisqualis (pre- and post-harvest), one application of mineral oil before bud break, and model-based applications of sulphur fungicides between bud break and fruit set.

Blue light acts as a double-edged sword in regulating sexual development of Hypocrea jecorina (Trichoderma reesei).

The industrially important cellulolytic filamentous fungus Trichoderma reesei is the anamorph of the pantropical ascomycete Hypocrea jecorina. H. jecorina CBS999.97 strain undergoes a heterothallic reproductive cycle, and the mating yields fertilized perithecia imbedded in stromata. Asci in the perithecia contain 16 linearly arranged ascospores. Here, we investigated H. jecorina sexual development under different light regimes, and found that visible light was dispensable for sexual development (stroma formation and ascospore discharge). By contrast, constant illumination inhibited stroma formation, and an interruption of the darkness facilitated timely stroma formation in a 12 h/12 h light-dark photoperiod. The results of genetic analyses further revealed that H. jecorina blue-light photoreceptors (BLR1, BLR2) and the photoadaptation protein ENV1 were not essential for sexual development in general. BLR1, BLR2 and ENV1 are orthologues of the conserved Neurospora crassa WC-1, WC-2 and VVD, respectively. Moreover, BLR1 and BLR2 mediate both positive and negative light-dependent regulation on sexual development, whereas ENV1 is required for dampening the light-dependent inhibitory effect in response to changes in illumination. Comparative genome-wide microarray analysis demonstrated an overview of light-dependent gene expression versus sexual potency in CBS999.97 (MAT1–2) haploid cells. Constant illumination promotes abundant asexual conidiation and high levels of hpp1 transcripts. hpp1 encodes a h (hybrid)-type propheromone that exhibits features of both yeast a and a pheromone precursors. Deletion of hpp1 could rescue stroma formation but not ascospore generation under constant illumination. We inferred that the HPP1-dependent pheromone signaling system might directly prevent stroma formation or simply disallow the haploid cells to acquire sexual potency due to abundant asexual conidiation upon constant illumination.

Inoculum and disease dynamics of circular leaf spot of persimmon caused by Mycosphaerella nawae under semi-arid conditions

The epidemiology of circular leaf spot of persimmon, caused by Mycosphaerella nawae, was studied in a semi-arid area in Spain for two consecutive years. No conidia were observed on diseased leaves and all infections were thought to be caused by ascospores formed in the leaf litter. Ascospores were released mainly in April and May, but relatively low numbers in June were able to induce severe symptoms on trap plants. Temperature was not significantly correlated with ascospore catches or disease incidence on trap plants, indicating that it was not a limiting factor for disease development during the period of study. Rainfall was above normal, but still considerably lower than in endemic areas of Korea. Most infections coincided with rains, but the disease was observed also on trap plants exposed to less than 1 mm of precipitation and even in the absence of rain. Orchards were flood irrigated once inoculum deposits in the leaf litter had already been depleted, so it was not possible to determine its effects on ascospore release and disease development. The use of a wind tunnel to determine inoculum production allowed detection of physiologically mature ascospores of M. nawae in the leaf litter 1–2 weeks before they were released to air in the orchard. Disease progress was fitted to the monomolecular growth curve, associated with monocyclic pathogens and diseases with a variable incubation period as a function of the host phenology.

The effect of winter weather conditions on the ability of pseudothecia of Leptosphaeria maculans and L. biglobosa to release ascospores

Leptosphaeria maculans and L. biglobosa are damaging pathogens of oilseed rape. The infection of plants occurs predominantly in early autumn or spring by spores produced in pseudothecia. The aim of this study was to investigate whether pseudothecia formed in the autumn are still viable in the spring and to what extend they are destroyed by winter frosts. The studies presented here demonstrated that winter frosts can render pseudothecia unable to release spores. Nevertheless, ascospores present in pseudothecia unable to discharge ascospores, were fully capable of germination, regardless of the incubation temperature. No significant differences were found between the studied Leptosphaeria species in their response to frost. A multiple regression equation has been elaborated to forecast the ability of pseudothecia to release ascospores, based on winter temperatures. Considerable correlation was found between the ascospore release in the autumn and the ability of pseudothecia to release ascospores over the winter period and the subsequent symptoms of stem canker before harvest. We have demonstrated that the potential and the survival of inoculum can have a large impact on the success of the pathogen. This may be particularly important in the light of forecasted climate change. Higher winter temperatures may increase the ability of pseudothecia to release ascospores and the discharge of ascospores of L. maculans and L. biglobosa into the air, and cause early plant infections. This in turn will increase the number of infected plants, the disease incidence at harvest, and reduce the yield of oilseed rape.

Sexual Development and Ascospore Discharge in &lt;em&gt;Fusarium graminearum&lt;/em&gt;

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in &lt;em&gt;Fusarium graminearum&lt;/em&gt;

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished). The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay. F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.