PDA (Potato dextrose agar) is a good medium for sporulation of phytopthora infestanse

Dear Yanuar,

Your point is really interested because the difficulty of culturing certain fungi particularly the Oomycetes Phytophthora infestans. The P. infestans treats as biotrophic pathogen in the early stage of its development and thus, it needs host tissues to make it able to germinate and sporulate. So, I advice you to use the whether potato slides and/ or Rye extract medium. You can also use these two protocols in the same in order to save time and to get results. With potato slides, you should bring potato tubers, wash them under tap water and then you apply surface sterilization with any sterilized solution for not more than 5 minutes. Cut them into slide with good thickness and then apply again surface sterilization for not more than 2 minutes. Place each slide in a sterilized Petri dish, and don't forget to put a witted sterilized filter paper under each slide. Inoculate each slide with small inoculum and incubate the plates on 27 C in the dark. Check the development of P. infestans using bi-nuclear and you will get on very clear sporangia and sporangiospores.

For, The Rye medium, bring up about 200 - 300 g rye seed. wash them using tap water and them soak seed for a while. Add more water and then boil seed for a while. Extract seed and use only extraction after adding 20 g sucrose and 20 g agar and then complete it to 1 L. Autoclave and go forward. You can also using bean extract medium protocol. It would give good results

For, Alternaria solani, the fungus is quiet easy to grow on artificial medium, I suggest you to use Faba bean (Vicia faba) extract medium rather than PDA. However, PDA is really limited growth medium and don't forget that fungi needs selective medium. Bring 200 g faba bean leaves, wash them in tap water and boil leaves with water for a few minutes ..not for long time. Don't reach to the boiling point because the leaves have oxidants and they will turn into be dark. Discard leaves and use extraction, add 20 sucrose and 20 g agar. Autoclave on 120 C for only 5 minutes. and then go forward

Alternaria = PDA as well, agar concentration 2%, for PDA mixture: I use 8 g for 200 ml of medium. PDA you may prepare yourself from fresh potato (cheap). I never multiply Phytophtora, Thank you Gajjan for your valuable advice. I use Alternaria and Botrytis cultures for the plant parasitic nematodes multiplication: Aphelenchoides, Bursaphelenchus (bulb and leaf nematodes). Phytophtora may be good to multiply the nematodes of the genus Ditylenchus (stem nematodes).

Maybe you can try a oatmeal medium. It`s a good medium for macrospore production.

For Phytophtora V8 agar is best media but should be supplemented with antibiotic like Vancomycin and Temp below 20C as it is a slow growing fungi

Yes PDA (Potato dextrose agar) is a good medium for sporulation of these fungi, because I already used this medium to cultivate both fungi.

I have used tomato juice extract agar for Alternaria solani sporulation. First cultivate the fungus on PDA plate and when you get actively growing mycelial mass transfer it to tomato juice extract agar plates and make sure to incubate it in dark other wise it wont sporulate. For Phytopathora try PDA prepared with freshly boiled potatoes rather than premixed PDA supplied by the manufacturer.

it works really well i have used it for Botrytis, Phytopathora, Macrophomina. Worked well with all.

V8 medium is very good to Alternaria species in general at 20 C, while potato tuber was the best to sporlation Phytophthora infestant at 13-18 C.

Hey I have worked with both organisms and they sporulate very on V8 medium. in dark condition and temperature around 25C. it will take up to 2 weeks to have a very good sporulation. PDA also works for both, it takes longer however.

Potato is best for the sporulation of Phytophthora infestans and leaf extract agar medium for Alternaria solani.

Yanuar, some of your isolates maybe asporous mutants, try standard fruiting isolates first. For example I use asoprous Botrytis to culture fingal feeding nematodes on it

Some years back I red an article according to that , keeping the plates of grown mycelium of A. solani on sun light 10 minutes /day for 3-5 successive days promotes its abundent sporulation and I did it

my experience in inducing sporulation of Alternaria is by cutting the medium that has fully grown by the fungus. In the cutting area, we can observe the conidia. For Phytophthora infestans, I also used a cut potato tuber placed to grow a slice of fungal culture and stored in refrigerator. After 1 weeks, sporangia of the fungus can be collected by dislogging them with aquadest.

Hi Yanuar,

Make sure that you do get Phytophthora spp.

For Phytophthora spp isolations, a sharp scalpel was used to cut small (1 mm) pieces of infected tissue that were transferred to plates of modified selective CMA–NARP (17 g/L corn meal agar [Difco], amended with 1 mg/L nystatin, 100 mg/L ampicillin, 100 mg/L pentachloronitrobenzene (PCNB) and 10 mg/L rifampicin) (Shearer & Dillon, 1995). Plates were incubated at 20 degree C for 10 days.

To induce the production of fruiting structures, isolates were grown on either CA or V8 agar (354 mL V8 juice, buffered with 5 g of CaCO3, 15 g of agar, 1 litre with distilled water) (Erwin & Ribeiro, 1996). For sporangia, blocks (5 × 5 mm) were cut from the edges of actively growing 7-day-old CA and V8 cultures, and immersed directly into non-sterile soil water (Erwin & Ribeiro, 1996), with modifications (60 g soil in 1 litre of water, allowing the soil to settle for 8 h and filtering the supernatant through four layers of cheese cloth). Blocks of agar bearing mycelium in the soil-water mixture were incubated at room temperature (20–22°C) for 12 hrs and then transferred to a 4°C incubator for 4 hrs. Cultures

were then returned to room temperature and inspected every 30 min for the presence of fruiting structures. In an attempt to produce oogonia, pure cultures growing in CA and V8 were incubated in the dark at 20°C and 25°C and

checked at 14 and 30 d. Isolates were also paired with each other in all combinations on the same media and under the same incubation conditions, and observed at 14 and 30 days.

You can keep the sealed plates containing mycelia of Alternaria solani under sun light for 10 minutes for 3-5 days . It will promote its sporulation

There are many methods reported in the literature that have worked, but for 1-2 isolates. In my experience, dealing with dozens of Alternaria solani isolates (good to see differences between A. solani and A. tomatophila, according to Simmons) concluded that some do not sporulate ever. I suggest: i) use another isolated, or ii) using mycelium as inoculum, it is equally effective both in vitro and in the field (if inoculas in the afternoon) or iii) continue testing methods.

Simmon argued thar A. solani is from potato and sporulate well; but A. tomatophila is the Alternaria from tomato an sporulate barely

Thanks very much for all of your advices!

czapaks Dox agar medium is best media for Alternaria solani and V8 juice medium for Phytophtora infestans

Phytophthora is sporulate in freshly harvested rain water, simply collect freshly rain water in tube and inoculate the mycelium of Phytophthora and after 4 to 7 days sporulation take place.

i did work on citrus Phytophthora spp. from long time, in view phytophthora sporulation on isoaltion of soil or other that required specific incredient after that cut the 7mm pieces on medium where phytophthora colony grew and add simple distilled sterzd. water and add in petriplates and see sporulation other sorghum grain was also very good medium

In many recent papers, they induced sporulation by incubating for 6 days at 23–25°C on PDA under near-ultraviolet light (310–400 nm) with 16 h of daylight. It seems like working. Im about to try this too

Some of these methods, mentioned in the string, do not favour release of zoospores from the sporangia. Can anyone share his/ her experience on that?

For Inducing sporulation in Phytophathora use Charcoal water (20 gram charchoal+ one litre water and for sporulation in Alternaria use Alfalfa seed decoction (Alfalfa seed-20 g. +water 0ne litre or PDA

If you have isolated Phytophthora infestans successfully (we pick sporangia from infected potato leaves onto antibiotic rye agar using agar cubes avoiding touching the leaf surface as far as possible - for media recipes see the Eucablight network at www.eucablight.org) then it should sporulate on any of the standard media used for it e.g. rye agar, pea agar, carrot agar. It doesn't need light or anything fancy to make it produce sporangia. We find most profuse sporulation on pea agar, but the cultures must be really clean i.e free from bacteria. For zoospore production, sporangia can be washed off the agar cultures using sterile distilled water and stimulated to release zoospores by chilling for 1-3 hours at 5-10°C then allowing the suspension to warm to room temperature. Aged sporangia from older cultures will be less inclined to release zoospores. You can check you have isolated P. infestans by inoculating it back onto detached potato or tomato leaflets (put drops of sporangial/zoospore inoculum on the leaf surface) and looking for infection and production of the characteristic lemon-shaped sporangia (which are also easily recognised from agar cultures).

I have similar problem on culturing A. solani from infected tomato leaf . I tried to use both V8 and Corn Meal Agar, still don't work .

Corn Meal Agar should enhance sporulation of Alternaria solani

Has anyone used Potato Dextrose agar for culturing frozen ATCC P. infestans? Otherwise, could you suggest a working protocol?

Thanks! 

has anyone done seed inoculation for a soil borne fungus before,

For Alternaria solani see Rodrigues et al 2010. In vitro production conidia Alternaria solani. Trop. Plant Pathol. 32(4):203=212.

Light stimulation is critical.

For Phytophthora infestans use tuber and fruit slices to bait isolating off young lesions.

In addition to Paul's suggestion, I found another newer article for Alternaria. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.828.4511&rep=rep1&type=pdf