I've tried growing it on a fruit, on leaves and brushing an agar plug but no success yet. I have tried putting the sporangia directly on tomato leaves, fruits but they rot before any P. infestans grows?
Be aware that potato races may be (usually are) less virulent on tomato (and vice versa).
In an aseptic laminar flow hood, we surface sterilized tomato leaves in 10% bleach solution for 5 min, then rinsed them twice in ddH2O for 5 min each, and then inoculated them with ~20 ul droplets of zoospore/sporangia suspension at ~10^4-10^5 cells per ml. We incubated them in autoclaved plastic boxes lined with moist, sterile filter paper at 17C, and usually saw symptoms in a week or so, depending on pathogen isolate and host genotype. It is very important to maintain high humidity in the boxes, so we would wrap the edges with parafilm and/or place the entire box into large ziplock bags.
Once fluffy mycelium is visible on the leaf surfaces, you can transfer some to antibiotic plates (e.g., PARP or RVN, we used RVN) and then subculture to ensure the purity of the isolation.
A lot of these steps were necessary to maintain our pure isolates. If you just want to culture some random P. infestans, you can do the same thing with field-collected infected leaves. Just put them in a plastic bag and incubate them for a day or two until they get fuzzy, then plate the mycelium on antibiotic medium. Subculture to purify the isolate, then run PCR (or Koch's 3rd and 4th postulates) to confirm that what you isolated is actually P. infestans.
I've linked one of our pubs with detailed materials and methods. Best of luck with your inoculations!
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464107/
I agree with the protocol described by Dr. Jack Erron Haggard.
Best wishes.
Dr. Mostafa Helmy Mostafa
As Jack has adviced, Pi usually forms abundant sporangiophores and sporangia on the surface in moist chamber within 1-4 days at 17-20 degrees if the symptoms are fresh.
I would just like to add this additional information: if you happen to have problems with (bacterial) contamination (even on selective medium), you can try to put infected leaves or sporangiophores/sporangia between air-dried potato tuber slices (ca. 0.5-0.8 cm thick) or in a hole made with cork borer in a clean and surface desinfected young tuber of a susceptible cv. Keep the slices in moist chamber at 20 C degrees with no contact between the slices and paper towel (mycelium will grow through the slices and you can easily take it from the surface). Regarding the whole tuber, you can slice it when symptoms develop near the inoculation point or wait until you see sporangia on the surface.
Good luck,
József
Dr. Anne Wathoni Njoroge,
There is another protocol which need stored potato tubers ( at least for 6 months), sterilize them, then using sterilized scalpel cut the tubers crossing , but do not separate the portions. Take a piece from infected tomato leaf and put it between the portions. close the portion and encase the tubers by grayed paper. Incubate tubers at 18 C for three days. You would found that the pathogen infected the tubers, then you can isolate on suitable medium the pathogen. After that you have to take sporangium in sterilized distilled water in strilzed tube. Incubate glass tube containing suspension at 4-6 C for one hour, than you can get zoospores. Inoculate tomato leaves from the lower side by drops of zoospores. I hope that you can accomplish your mission.
Dr. Mostafa Helmy Mostafa
Dr. Mostafa,
Thanks a lot. We have host specialization on potato and tomato. Isolates on tomato don't infect potato. I have tried to grow mycelia from tomato on potato tuber slices but they failed to grow. I don't know if the stored tubers will be any different but i can give it a try.
(1) Sato at al. 1991 A Newspaper Bag Method for Sample Collection of Blighted Potato of Phytophthora infestansLeaflets for Isolation. Ann. Phytopath. Soc. Japan 57: 573-576
https://www.jstage.jst.go.jp/article/jjphytopath1918/57/4/57_4_573/_pdf
(2) LABORATORY MANUAL FOR P. INFESTANS WORK AT CIP-QUITO
http://documents.plant.wur.nl/pri/Biointeractions/Lab%20protocols_PLB/CIPmanual97.pdf
(3) Isolation of P. infestans from infected tomato leaves
https://research.cip.cgiar.org/typo3/web/index.php?id=325
(4) J. Tumwine 2000 Isolation techniques and cultural media for Phytophthora infestans from tomatoes Mycologist Volume 14, Issue 3, August 2000, Pages 137–139
http://www.sciencedirect.com/science/article/pii/S0269915X00800968
Hello Anne
You get plenty of inoculum for this fungus by taking about one cm away from the edge of fungal colony of Phytophthora and put it in petri dish containing distilled water plus leaf discs of potato or tomato, after 24 h you can get good sporulation and sporangium as well. Pease see the attached references:
Good luck
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