The simplest way is to filter spores from fragments of mycelium made by macerating actively growing cultures. The best filter to use depends on the size of spores but typically a few layers of sterile muslin can be used, otherwise you will need to use a membrane with a known filtration threshold. Normally we encourage sporulation by placing cultures under nearUV light, once the colonies have established.

Lucia,

which fungus are you working with?

I don’t know what you need the spores for, but if you filtrate the spores you need to have them in some kind of liquid which may affect them. If it important not to affect the spores by a liquid you can expose the fungal culture to a clean airflow and then during the air exposure sample the aerosolized spores on a filter using e.g. a Millipore sampler or GSP sampler.

It normally depends on the type of fungs you are working with, the best way is to change the growth media and find a media that will favor the growth of spores (excluding certain nutrients from media can stimulate sporulation). What also helps is changing the cultivation conditions as some temperatures will encourage sporulation.

These things are involved: Filteration or airflow, Selective media which depends on the fungi spore of interest, UV light and good antiseptic technique.

It depends on the fungus that is working. I agree with the answer, I believe also that he should seek a culture medium supplemented with or lacking some nutrient that promotes the growth of spores.

Growth until the fungus assimilates all the media...after that the fungus will produce the spores...

As well as growth media, inoculum density can make a difference for some species, with overcrowding on a plate leading to more hyphal growth. You can try different dilutions, or just try streaking out the inoculum rather than spreading over the whole plate.

It's so simple ! But depends on fungus species. Culture the fungus in tubes, on slant agar and incubate at appropriate temperature. When sporulation is maximal, add several mL of 0.1% Tween 80 in saline solution to eluate the spores which are on the upper side of the colonies. Use a loop to grab the spores or a pipette to aspire the suspension.

Several answers, all correct, but without knowing what the fungus is difficult

Again, agreed. The genus is important to know. I work on Cercospora and it sporulates on V8 media.

You didnt mention which fungus and for what you need the spores. I agree with the answer of Ntsoaki Malebo. Thiis is the method to induce sporulation in fungi, nutrient dificient media such as Czapek agar usually encourage sporulation.

Spore production is largely increased upon starvation, just wait until nutrient supply is exhausted

Try culturing on minimal medium (like tap water agar). Good luck!

I am working with Trichoderma. It forms spores frequently when we put it at higher temperature and in light. minimum nutrient availability also increases spore formation but conditions may be different for different fungal species.

Dear Lucia. You can let growth your fungus in solid medium containing salt ( 10mM to 100mM of NaCl or Cacl2) and at normal growth temperature growth for 3 to 5 days. They will sporulate very well.

Good luck

We are working with Alternaria

You can tray oat meal agar: 20g oat meal per liter. Heated for 30 min to 1h, separate de solid and add 1.5 - 2.0% agar. I used various fungi. Good luck

There's more than one way to skin this cat (apologies cat lovers!)

1. Oatmeal and cornmeal agars enhance spore production in most filamentous organisms, both fungi and bacteria.

2. You can place sterile filter papers on agar plates and inoculate on top of the paper. After growth you can slowly peel the paper off the media picking off the spores.

3. You can pour Tween 80 or 0.2% SDS (this reduced hydrophobicity of spores) on fungal colonies, make a suspension and filter it through sterile cotton wool. Cotton wool traps hyphae which are long and lets circular spores to pass through.

4. If you need to remove spores and be quantitatively precise - remove a block of fungal colony, place it in 0.2% SDS solution (in a falcon tube) and shake it overnight. This will slowly release the spores which you can quantify using a haemocytometer.

I have used all these methods in the following papers:

Antony-Babu & Singleton (2009) Effect of ozone on spore germination, spore production and biomass production in two Aspergillus species. AVL International Journal of General and Molecular Microbiology 96(4), 413-422

Antony-Babu & Singleton (2011) Effects of ozone exposure on the xerophilic fungus, Eurotium amstelodami IS-SAB-01, isolated from naan bread. International journal of food microbiology 144 (3), 331-336

The agar media culture like Zapec Dox are very good for sporulation, and you must wait antil media nutrients were depleted.

Lucia,

1. grow Alternaria on an agar medium (eg. potato-dextrose-agar or malt-extract-agar or others). Prepare a conidia suspension in distilled sterile water, and spread 0.5mL of this suspension on the entire surface of a Petri plate containing the agar medium; incubate at 22-25°C in the dark for about 1 week

2. harvest conidia: poor about 25 mL sterile distilled water onto agar surfarce of the colonized plate; handly swirl gently to favor detachment of conidia from conidiophores into the water; put the water in a flask, so you have a conidia suspension without hyphae (water can be previously added with 1-3 droplets/liter of Tween80 or Tween 20, Sigma, or other tensioactives to reduce hydrophobicity; also, instead of water, a physiological solution with 8.5g/L sodium chloride, 1g/L agar, 1-3 droplets/L Tween 80 or Tween 20 can be used to avoid conidia lysis)

Higher level of salt, (NaCl, CaCl), Lower Sugar and very useful is put it under the UV light.

sometimes helps mechanical removing mycelium from the agar (let it grow firstly as a mycelium on the whole dish). After it remove mycelium with a little amount of sterile water and loop. Then you put it to dark (or try UV) or both. Only conidiophores then are rising directly from agar after two or more days.

I agree with the above suggestions. Some fungi sporulate better under near UV light.

better way to use Tween 80 solution (0.1%) for separation of spore from fungal hyphae

I agree with Bart. For example, elevated CO2 inhibit microsclerotia production n Verticillium dahliae. I have not information about Alternaria. However, a simple test may ascertain it.

In case if you want to have more spores, inoculate the culture on autoclaved seeds (pre-soaking in water for 6 hrs) of wheat or sorghum in conical flask, incubate at 28 degree Celsius in dark. Some fungi require light or NUV light for sporulation. Most of the laboratories use this method to prepare spore inoculum tospray on germ plasm lines for testing resistance to that pathogen.

I agree with Sanjay Antony-Babu in the four ways mentioned. The use of UV light is also encouraged including media selectivity under good aseptic conditions

latest publication on the subject was published in the journal Mycology 2012,V.3, 3, 195-200 Induction of sporulation in plant pathogenic fungi - good collection of methods

Inflicting physical injuries (with sterile needle) on young mycelia in minimal media would make the organism become hardy and stimulate early and copious spore production. other suggestions could also accompany this one in various combinations. Note that formation of sexual apparatus is also encouraged under this harsh conditions.

you amend your medium with host extract where from fungs isolated or can put some sterile carnation/or banana leaf pieces on Petri dish and I think it may help the induction of spore proliferation. RNK

the most simple way you can do is to just minimize the amount of nutrition in the media, e.g. if you are using PDA then decrease the amount of glucose, some fungi can even just grow on the simple agar media as well, even though minimizing the agar concentration of agar will also increase the possibilities of induction of spore production.

Allow aerial growth , if possible adopt diphasic fermentation which will give your matured aerial spores in large quantity .

Growth on PSA (potato sucrose agar; instead of PDA) under will increase spore production in Fusarium app, particularly under UV. Worked very well for me anyhow and a routine method in the fungal lab where I worked at the time.

There may be early spore formation takes place when media is mixed with definite concentration of metabolites of certain antagonistic bacteria or fungi. I have earlier tried it for spore production in Trichoderma and the results are better.

From a very general point of view, the reduction of simple sugars (for example glucose) in a medium will help to reduce mycelium production in favour of spores. Good media are Malt agar, Oatmeal agar, Lima bean agar, V8 agar, but many other can be useful depending from the fungus you are working on. A good resource of recipes is the Difco Manual (http://www.bd.com/ds/technicalCenter/documents.asp). You should also pay attention to environmental conditions: for example some fungi require light to sporulate (continuous light or 12/12 alternating light/darkness), a very good source of light are the so called black lamps that irradiate Near UV light (NUV, 310-410 with a peak at about 360). The "diurnal sporulators" require a 12/12 alternating light/darkness with lower temperatures during the darkness period (for example some Alternaria). If your fungus doesn't yet sporulate you can try to damage the mycelium (thanks Zubbair !) by a scalpel or by burning the aerial mycelium by a Bunsen burner (be carefull !!).

To collect spores it is very important to reduce the amount of mycelium, than you can wash the plate and collect the spore suspension. Otherwise you can grow your fungus on a substrate of your choice overlaid by a cellophane sheet (see also point 2 from Sanjay Antony-Babu). When peel off the cellophane, spores (and mycelium) are easily collected free from the growing medium. To collect spores without the use of water, you could use a Cyclonic separation, but it could be quite expensive.

Try minimizing the nutrient content of the media used.

Some time stress condition stimulate sporulation for example short exposure of UV and Iodine solution...

Liquid culture in a flak on a shaker is desirable....After that you need to filter to collect the spores...

Use minimal media, usually v8 agar or alternatively change the temperature where you incubate, some fungi sporulate at lower temperatures.

Best thing you can go for a natural media from plants/fruits

Read something on Microcycle Conidiation. It's the induction of sporulation right away from new forming hyphae. 

Hi!

Some times the use of different light frequencies promotes sporulation. The lack of nutrients or the liquid media also use to promote sporulation.

Some time by some stress condition sporulation occur for example UV- lite treatments, by pouring Iodine solution solution in growing plate atc.

Exposure to NUV (near ultra violet) light enhances sporulation in fungi like Phomopsis vexans, Colletotrichum spp., a kind of stress.