Hi, You can not isolate downy mildew on artificial medium because it is a obligate parasite, so you should do all the steps on live plant.

First make very diluted spore suspension and inoculate the susceptible host and incubated in moist condition to get infection on leafs. Try to collect spores from single spot infection on leaf that theoretically came from single spore and re inoculate susceptible host to increase it, that will give you a pure culture of the pathogen.

To identify it you can use the morphological characterization of the sore and conidospore shape depend on the pathogen hosp, there are a special keys for each genius of downy mildew pathogen. Or you can use molecular techniques.

As Oadi Matny has stated biotrophic pathogens such as downy and powdery mildews are not cultured in the laboratory on artificial media. The identification of these fungi is mainly based on the spore morphology and their arrangements and as Oadi Matny suggested molecular techniques can be useful. Your experimentation can use susceptible spreader rows to experiment with cultivar resistance differences. Fungicidal agents can be used on susceptible varieties and the disease severity and damage evaluated based on this relationship. In crops such as soybeans single leaflets can be rooted in sand culture and the powdery or downy mildew kept in laboratory in sterile cultures under controlled growth environments.

Definitely like all obligate parasites, you can not culture Downy mildew in artificial media. You can however create spore suspension in water from infected material/leaf and and then inoculate the susceptible test plant and observe for symptom development

Hey guys,

Thanks for the feedback. I'm aware that its not culturable thus the questions. Glad to hear that a single colony produced on a leave can be used as a pure culture for reinoculation and confirming the disease. I'll give it a try.

Thanks again,

Pieter Louw

1. What specifically is the native crop?

2. What downy mildew pathogens have previously been reported on it, or a species in the same genus, or at least a related genus?

3. Once you have answered the previous two questions, I think a simple literature search should suffice to help you find inoculation methods. Most DMs are foliar pathogens, so you "merely" need to collect some viable sporangia from infected field material, spray it on your healthy test plants, keep them in an environment to have dew and the appropriate temperature for 12+ hours, and then wait for symptom expression.

4. My experience is with Plasmopara halstedii on sunflowers, which is the oddball DM in that it infects via the roots, specifically with seedlings, and induces a systemic infection. Very, very few mildews operate that way.

I'd be interested to hear the answers to the first two questions.

Sincerely.

Hi Dr Gulya,

I'm helping a postgraduate student and according to him nothing has been reported. I still need to look into it more myself but will only find the time next week. From the little I do know and looked at, I can't find any publications associating the disease with the crop. All I can say now is that it's an experimental pharmaceutical crop. I'll have to check back with you when I know more.

Appreciate the interest,

Pieter Louw

Dear Dr Gulya,

The fastest and easiest method to confirm pathogenicity would be by using the in vitro detached leaf assay. Would you advise this and on what media would you suggest? I'm guessing it depends on the host and pathogen or will water agar suffice?

Kind regards,

Pieter Louw

If you can root the detached leaves they can be maintained in sterile sand which is irrigated with Hoaglund nutrient solution. The cultures can be maintained in a growth chamber with 12 hour light and dark and moderate diurnal temperatures. Downy mildew will need to keep with a film of water of enough hours to ensure the penetration and establishment.

Is it possible that some powdery mildews (i.e. Phyllactinia guttata) only grow on the underside of the leaf?

The detached leaf assays were developed by Mignucci and Lim for soybean powdery mildew culture. It has used in IITA for soybean rust with success. Check out the references they should be useful for you.

Yes Phyllactactinia guttata is common on the leaf underside. The telemorphic state has black hemicircular cleistothecia. The appendaes are sword shaped and will position the cleistothecia according to changes in humidity. The conidia are terminal in a chain and are hyaline. The pathogen has a broad host range. Beside the morphological identification molecular techniques could be employed to confirm the identification of your pathogen. While downy mildews require substantial free water for leaf penetration powdery mildews are not so moisture dependent.

Then I'm starting to think the pathogen is Phyllactinia guttata or at least a related species (see attached). If molecular techniques are used (I prefer it), the only genomic data I could find is on the ITS or 18S rRNA, non of which is specific enough for species identification. I guess morphology has been adequate in the past?

If you have healthy young leaves you can put them in a vial of water with a cut petiole. The leaves may sprout from the excised petioles which then these can be use for culturing the fungus. The conidiophores can be scraped and put on a healthy excised leaf for culturing. In this pathogen the information is the infection is on the backside through leaf stoma. If you use a piece of clear cellophane you can peel off the conidia and conidophore the anamorphic stages Ovalariopsis I believe. The microphotographic evidence would differentiate the powdery mildew from a down mildew stage. I think you are probably on the right track that this is a powdery mildew. Late in the season is when the telomorphic stage is produced. Clestothecia are quite interesting and distinctive.

there a re some older papers of Dai et al 1994, where are a lots of parctical thing including hstochemistry

If you are suspecting as the photos seem to indicate Phytlactinia guttuata this type of of diasese is a powdery mildew of a asocmycete affinity and tnot a downy mildew of a Oomycete affinity.

The proper identification can be confirmed by morphological characters of the asexual and sexual stages clearly.

Genomic DNA based techniques can give added confirmation.

The Powdery mildew cultures can be maintained in tissue cultured plantlets in axenic culture.