Name: Sexual Development and Ascospore Discharge in Fusarium graminearum (Video) | JoVE Journal
Uploaded: 2012-03-29T13:00:00.000+00:00
Duration: 8 min 20 s
Description: Learn techniques for inducing sexual development and assaying spore discharge in Fusarium graminearum. This method enables genetic crosses, recombinant progeny evaluation, and mutant screening for fungal biology research.

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5 minutes

This work was supported by a grant from the National Science Foundation (MCB-0923794 to FT).

1. Producing Perithecia
<ol>
	<li>Center inoculate carrot agar10 in a 6.0 cm diam Petri dish with a fertile strain of F. graminearum (Recommend PH-1).</li>
	<li>Place inoculated dishes under bright fluorescent lights at room temperature (18-24 °C) and allow to grow until mycelium has reached the outer edge of the Petri dish (3-5 days). Commercial household fluorescent lights work fine for stimulating fruiting body formation and discharge.</li>
	<li>Gently remove the aerial mycelia with a sterile toothpick.</li>
	<li>Distribute 1.0 ml 2.5 % Tween 60 aq to the surface with a sterile glass hockeystick or rounded end of a sterile glass rod.</li>
	<li>Return plates to light. Do not add Parafilm to plates.</li>
	<li>After 24 hr, the surface of the plates should have a shiny appearance. If mycelia reappears, scrape surface and re-apply Tween-60 solution.</li>
	<li>Observe development of perithecia over the next week. The intermediate stages of perithecium development can be harvested by gently scraping the surface of the agar and flash frozen for RNA extraction (Figure 1).</li>
	<li>Mature spores will accumulate on the lid of the plate by day 7. Initially these will only be visible under a dissecting microscope, but by day 9, they will have accumulated to sufficient density so as to be visible to the naked eye.</li>
</ol>
2. Ascospore Discharge Assay
<ol>
	<li>On day 6 after application of Tween solution, cut a 1 cm diam circle out of the agar with a wood borer. Alternatively, a scalpel can be used to remove a similar sized segment. The circle can be sliced in half and each half placed on a glass microscope slide.</li>
	<li>Orient the pieces so the surface containing the fruiting bodies is perpendicular to the surface of the slide, and the spores are fired down the length of the slide.</li>
	<li>Place the slide in a humidity chamber overnight under lights. Spores will accumulate on the slide and be visible to the naked eye the next morning (Figure 2).</li>
	<li>Accumulated spores may be washed off the slide with water and quantified if desired.</li>
	<li>Potential ascospore discharge inhibitors can be assayed by adding them to the back side of the agar block during assembly of the assay. Some ion channel inhibitors that reduce discharge have been previously assayed with this technique15.</li>
</ol>
3. Generation of Recombinant Progeny
<ol>
	<li>Initiate cross by inoculating carrot agar with two strains of F. graminearum (one Nit+ and one nit-), placing the inoculation points on opposite sides of the Petri dish.</li>
	<li>Once hyphae have filled the surface, scrape off aerial portion and apply Tween-60 solution as described above. Use a marker to note on the side of the Petri dish where the hyphae meet.</li>
	<li>Return cultures to light and incubate until perithecia have developed, and cirrhi have begun to form from the perithecia along the intersection between the two cultures.</li>
	<li>Under the dissecting microscope, bring into view the perithecia along the intersection of the two cultures. Using an insect pin, sterilized and dipped into sterile water, touch a cirrhus along this border lightly with the tip of the pin. The moisture on the pin should allow the cirrhus to stick to the pin.</li>
	<li>Rinse the tip of the pin with the cirrhus on it in 200 μl sterile water in an Eppendorf tube to wash the spores off.</li>
	<li>Flame the pin, moisten it again and pick another cirrhus. Pick 10 to 15 cirrhi, and place each in a separate tube of water.</li>
	<li>Briefly vortex tubes containing suspended cirrhi.</li>
	<li>Remove 60 μl of the spore suspension with a pipette and place on MMTS medium1 in a 9 cm Petri dish. Spread with a sterile hockey stick. The remaining spore suspension may be stored at 4 °C for up to a week and replated if necessary.</li>
	<li>Incubate at room temperature (light not necessary) for 3-5 days until very small colonies appear (Figure 3). There will be two kinds of phenotypes among the colonies. The nit- colonies will have sparser hyphae than the Nit+ colonies. Cirrhi from recombinant perithecia will show both colony phenotypes in approximately equal proportion. Discard the plates with colonies of only a single phenotype on them. Note that sometimes more than two phenotypes result due to segregation of other factors.</li>
	<li>Move colonies to V8 or carrot agar for storage. Test colonies on Czapek's Dox Agar (the nit- strains will not grow on Czapek's Dox) and on PDA with Chlorate (0.5 M Potassium Chlorate; the Nit+ strains will not grow on PDA with Chlorate) to confirm the correct phenotype. Examine these colonies for the desired qualities.</li>
</ol>
4. Representative Results
Producing perithecia
The goal is to have a lawn of perithecia without any mycelia or spores apparent. The surface of the culture will appear similar to black velvet (Figure 1). At 24 hr after the application of Tween the surface of the agar should have a slight sheen. If mycelia reappear, then the plate may not have been scraped enough to induce perithecium development.
Ascospore discharge
Always provide in your assay several blocks of agar from the wild-type strain. If those do not fire, then either your perithecia are too young or too old. If they are too old, then numerous hyphae will appear over the surface of the culture giving it a whitish hue. If this is not the case, they may be too young, and the assay should be tried again in 24 hr. Occasionally, cultures do not discharge well, and the experiment will have to be repeated. Be sure you perform the assay under the proper light conditions.
Recombinant progeny
Recombinant perithecia should make up the majority of perithecia along the intersection between the two strains. We usually process 10 cirrhi from a cross and at least 4-6 of those should be recombinant. Very occasionally, a mutation prevents a strain from mating. If one of the parents has an aberrant growth phenotype, then more than 2 phenotypes may be present among the colonies on MMTS.
<img alt="Bacterial growth pattern analysis in petri dishes; fungal growth experiment result." src="/files/ftp_upload/3895/3895fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Stages of perithecium development on carrot agar. Left, 72 h after application of Tween. Note red pigment and very young perithecia visible as a black grains on the surface. Right, at 144 h, mature perithecia have formed. Note near-complete absence of superficial mycelia in both cultures.
<img alt="Fungal growth observation on agar; petri dish experiment; spore formation and spread analysis." src="/files/ftp_upload/3895/3895fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Discharge assay. The orange carrot agar plug is oriented so that spores forcibly discharged from the mature perithecia on its surface can accumulate on the surface of the glass slide. 15 hr accumulation time.
<img alt="Static equilibrium diagram with clusters; microscope image for sedimentation analysis, particle study." src="/files/ftp_upload/3895/3895fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Differences in growth between Nit+ and nit- colonies on selective MMTS medium. The nit- colonies (arrowheads) are smaller. The Nit+ colonies (arrows) show robust growth. Photograph taken after 7 days.

<ol><li>Bowden, R. L., Leslie, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexual+recombination+in+Gibberella+zeae%5BTitle%5D)+AND+%22Phytopathology%22%5BJournal%5D)">Sexual recombination in Gibberella zeae.</a> Phytopathology. 89, 182-188 (1999).</li>
<li>Buller, A. H. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBuller%2C+AHR+%22Researches+on+Fungi%22">Researches on Fungi</a>. , Longmans, Green and Co. London, England. (1909).</li>
<li>Cavinder, B., Hamam, A., Lew, R. R., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mid1%2C+a+mechanosensitive+calcium+ion+channel%2C+affects+growth%2C+development%2C+and+ascospore+discharge+in+the+filamentous+fungus+Gibberella+zeae%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascospore discharge in the filamentous fungus Gibberella zeae.</a> Eukaryotic Cell. 10, 832-841 (2011).</li>
<li>Cuomo, C., Güldener, U., Xu, J. R., Trail, F., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Fusarium+graminearum+genome+reveals+a+link+between+localized+polymorphism+and+pathogen+specialization%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization.</a> Science. 317, 1400-1402 (2007).</li>
<li>Desjardins, A. E., Brown, D. W., Yun, S. -H., Proctor, R. H., Lee, T., Plattner, R. D., Lu, S. -W., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletion+and+complementation+of+the+mating+type+%28MAT%29+locus+of+the+wheat+head+blight+pathogen+Gibberella+zeae%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Deletion and complementation of the mating type (MAT) locus of the wheat head blight pathogen Gibberella zeae.</a> Applied and Environmental Microbiology. 70, 2437-2444 (2004).</li>
<li>Gueldener, U., Seong, K. -Y., Boddu, J., Cho, S., Trail, F., Xu, J. -R., Adam, G., Mewes, H. -W., Muehlbauer, G. J., Kistler, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+Fusarium+graminearum+Affymetrix+GeneChip+For+profiling+fungal+gene+expression+in+vitro+and+in+planta%5BTitle%5D)+AND+%22Fungal+Genetics+and+Biology%22%5BJournal%5D)">Development of a Fusarium graminearum Affymetrix GeneChip For profiling fungal gene expression in vitro and in planta.</a> Fungal Genetics and Biology. 43, 316-325 (2006).</li>
<li>Hallen, H., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+L-Type+calcium+ion+channel+Cch1+affects+ascospore+discharge+and+mycelial+growth+in+the+filamentous+fungus+Gibberella+zeae+%28anamorph+Fusarium+graminearum%29%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">The L-Type calcium ion channel Cch1 affects ascospore discharge and mycelial growth in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum).</a> Eukaryotic Cell. 7, 415-424 (2008).</li>
<li>Hallen, H., Huebner, M., Shiu, S. -H., Guldener, U., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+shifts+during+perithecium+development+in+Gibberella+zeae+%28anamorph+Fusarium+graminearum%29%2C+with+particular+emphasis+on+ion+transport+proteins%5BTitle%5D)+AND+%22Fungal+Genetics+and+Biology%22%5BJournal%5D)">Gene expression shifts during perithecium development in Gibberella zeae (anamorph Fusarium graminearum), with particular emphasis on ion transport proteins.</a> Fungal Genetics and Biology. 44, 1146-1156 (2007).</li>
<li>Lee, J., Lee, T., Lee, Y. -W., Yun, S. -H., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shifting+fungal+reproductive+mode+by+manipulation+of+mating+type+genes%3A+obligatory+heterothallism+of+Gibberella+zeae%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae.</a> Molecular Microbiology. 50, 145-152 (2003).</li>
<li>Klittich, C. J. R., Leslie, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitrate+reduction+mutants+of+Fusarium+moniliforme+%28Gibberella+fujikuoi%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuoi.</a> Genetics. 118, 417-423 (1988).</li>
<li>Min, K., Lee, J., Kim, J. -C., Kim, S. G., Kim, Y. H., Vogel, S., Trail, F., Lee, Y. -W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+gene%2C+ROA%2C+is+required+for+proper+morphogenesis+and+discharge+of+ascospores+in+Gibberella+zeae%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">A novel gene, ROA, is required for proper morphogenesis and discharge of ascospores in Gibberella zeae.</a> Eukaryotic Cell. 9, 1495-1503 (2010).</li>
<li>Qi, W., Kwon, C., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microarray+analysis+of+transcript+accumulation+during+perithecium+development+in+Gibberella+zeae+%28anamorph+Fusarium+graminearum%29%5BTitle%5D)+AND+%22Molecular+Genetics+and+Genomics%22%5BJournal%5D)">Microarray analysis of transcript accumulation during perithecium development in Gibberella zeae (anamorph Fusarium graminearum).</a> Molecular Genetics and Genomics. , 276-287 (2006).</li>
<li>Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fungal+cannons%3A+explosive+spore+discharge+in+the+Ascomycota%5BTitle%5D)+AND+%22FEMS+Microbiol.+Lett%22%5BJournal%5D)">Fungal cannons: explosive spore discharge in the Ascomycota.</a> FEMS Microbiol. Lett. 276, 12-18 (2007).</li>
<li>Trail, F., Common, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perithecial+development+by+Gibberella+zeae%3A+a+light+microscopy+study%5BTitle%5D)+AND+%22Mycologia%22%5BJournal%5D)">Perithecial development by Gibberella zeae: a light microscopy study.</a> Mycologia. 92, 130-138 (2000).</li>
<li>Trail, F., Xu, H., Loranger, R., Gadoury, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+and+environmental+aspects+of+ascospore+discharge+in+Gibberella+zeae+%28anamorph+Fusarium+graminearum%5BTitle%5D)+AND+%22Mycologica%22%5BJournal%5D)">Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum.</a> Mycologica. 94, 181-189 (2002).</li>
</ol>

We have nothing to disclose.

F. graminearum is particularly well adapted to the study of fruiting body development due to the availability of a well-annotated genome (<a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/">mips.helmholtz-muenchen.de/genre/proj/FGDB/</a> ;4), and the availability of an Affymetrix-based microarray6. This has facilitated the ability to identify genes important for ascospore discharge7,11,3. The methods presented here will allow the researcher to focus on a discrete set of developmental stages and functions for genetic analysis of the fruiting bodies of F. graminearum. The methods are also easily adaptable to related fungi that can be induced to fruit in culture, and can be used as a standard for development in a variety of other fungal fruiting body types.
The ability to assess a spore firing phenotype can be subtle in species where spores are small and hard to see, such as F. graminearum. The collection of spores on a clean slide facilitates both visual assessment, and quantitative assessment, as spores may be washed off the slide and quantitated. Some fungal species produce a mucilage that surrounds their spore and the spores cannot be rinsed off, but must be counted microscopically as they remain on the slide. We have found this to be true of Sclerotinia sclerotiorum.

<table><tbody><tr><td>Fusarium graminearum strain PH-1</td><td>Fungal Genetics Stock Center</td><td>FGSC 9075</td><td></td></tr><tr><td>Tween 60</td><td>Sigma-Aldrich</td><td>P1629</td><td></td></tr><tr><td>Czapek&amp;rsquo;s Dox Agar</td><td>Difco Laboratories</td><td>233810</td><td></td></tr><tr><td>Potassium Chlorate</td><td>Sigma-Aldrich</td><td>255572</td><td></td></tr></tbody></table>

sexual development and ascospore discharge in fusarium graminearum

Sexual Development and Ascospore Discharge in Fusarium graminearum

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 crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

The overall goal of this procedure is to learn how to generate and manipulate parathesia development and ask APO discharge in culture. This is accomplished by first inducing and actively growing culture to initiate sexual development. The second step is to assay for active spore discharge, then recombinant parathesia are generated.The final step is to evaluate the progeny for phenotypes that indicate whether or not recombination occurred. Ultimately, the results can be used to screen for mutants in sexual development or to generate multiple mutations within one strain. Visual demonstration of this method is critical as the steps are hard to describe, and although simple, they must be done precisely to obtain optimal spore discharge and consistent parathesia production.Begin by center inoculating carrot auger in a six centimeter Petri dish with a fertile strain of f gramine. Arum incubate the inoculated dishes under bright standard commercial. Use fluorescent lighting at room temperature.Allow fungi to grow for three to five days until mycelium has reached the outer edge of the dish. At the hood. Gently scrape the surface of the culture with the sterile toothpick to remove the aerial mycelia.Then apply one milliliter of surfactant to the auger and rub into surface with the bent or bulbous end of a sterile glass rod. Without applying perfil. Place the plates back under the lights.After 24 hours, the surface of the plates should have a shiny appearance. If mycelia reappear res scrape, surface, reapply tween 60 solution and bathe them in light for an additional 24 hours. Now observe the development of the parathesia for a week.Young parathesia are visible as black grains on the auger surface. As compared to mature parathesia. There should be nearly no superficial mycelia.By day seven mature spores accumulate on the plate lid, and by day nine, the spores are so dense that they're visible without magnification. To harvest the intermediate stage, parathesia gently scrape the surface of the auger with a scalpel and transfer the parathesia to a cryo tube. The tube can then be flash frozen for RNA extraction six days after applying the surfactant.Cut a one centimeter diameter circle out of the auger with a wood, boer, or scalpel. Slice the circle in half and mount the halves on a glass microscope slide orient the pieces so the surface containing the fruiting bodies is perpendicular to the surface of the slide, and the spores are fired down the length of the slide. At this time, if an acro spore inhibitor is desired, apply it to the back of the auger block.This technique can be used to screen chemicals for discharge inhibitors and to screen genetic mutants for loss of the ability to fire spores, we have successfully done both. Now, incubate the slide overnight in a simple illuminated room temperature humidified chamber. As the fruiting bodies erupt, spores accumulate on the slide and will be visible there in the morning.Accumulated spores can now be washed off the slide collected in a clean dish and quantified. Begin by setting up a genetic cross between two strains of f gramine arum by inoculating them on opposite sides of a carrot auger petri dish. As previously described, grow the fungi until hyphie have filled the surface.Scrape off the aerial portions and apply T between 60 solution. In this scenario, mark the plate where the hyphie meet. Return the cultures to the light and incubate them for about seven days when the parathesia have developed and Siri begin to form from the parathesia along the intersection between the cultures.This technique can be used to combine multiple mutations into one strain. It's also worth noting that both knit plus and knit minus can be selected for making this a very useful selectable marker. Under the dissecting microscope, bring into view the parathesia along the intersection of the two cultures.Then find the sear eye. Now dip a sterilized insect pin into sterile water and touch the pin to a serous formed at the border between the cultures. The SRIs should stick to the pin's moisture.Then rinse the tip of the pin in 200 microliters of sterile water contained in a 1.5 milliliter micro centrifuge tube. Flame the pin, moisten it again, and pick another sru. Pick between three to five Siri and transfer them all to their own tube.Now briefly vortex. Each tube containing suspended Siri for each tube. Spread 60 microliters of spore suspension on a nine millimeter Petri dish.With MMTS medium, incubate the plates at room temperature with or without lights, and store the remaining spore suspension at four degrees Celsius for up to a week. After three to five days, very small colonies appear on the plate. One phenotype can be scored under a microscope.Knit negative colonies will have sparser hyphy than knit positive colonies. Discard those plates with only one of the phenotypes. The Siri from recombinant parathesia have both colony phenotypes in approximately equal proportion.Keep these plates. Sometimes other phenotypes result due to segregation of other factors such as colonies, which are intermediate between knit positive and knit negative for storage purposes, transfer the colonies from the recombinant plates to a V eight or carrot auger plate to continue the study by examining other phenotypes plate the colonies on Chap X dock's auger and on plates with PDA containing chlorate. Parathesia were grown into a black velvet like lawn without any mycelia or spores.24 hours after the application of tween, the surface of the auger had a slight sheen. If Mycelia had reappeared, the plate may not have been scraped enough to induce parathesia development. Several blocks of auger with fruiting bodies from a wild type strain were set up to fire their spores on glass slides to observe.Firing proper illumination was prepared. Parathesia that were too young or too old often did not fire. When too old, numerous hyphy appeared over the surface of the culture, giving it a whitish hue.When too young, the fruiting bodies did not fire, and the experiment was repeated. 24 hours later when 10 Siri were collected from a cross, at least six were typically recombinant as scored by a mix of knit negative and knit positive phenotypes. Very occasionally a mutation prevented a strain from mating.If one of the parents had an apparent growth phenotype, then more than two phenotypes were present among the colonies on MMTS. When attempting this procedure, it's important to remember to start with a culture that has been well-maintained and not sequentially transferred over time. Always start with a culture that's been grown from a stable freezer stock.

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Sexual Development and Ascospore Discharge in Fusarium graminearum

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