Hello

The best method I know of is deep-freezing in liquid nitrogen. However, this requires very clean cultures ( if your stock is contaminated by bacteria, these will invariably resume growth before the Phytophthora does once you take the samples out to culture them back again), and a slow freezing process for the first few steps ( down to -40 °C approx). The contamination issue is especially crucial for P. infestans, which is a strict biotroph in nature and grows well axenically only on rich media, where bacteria strive as well.

Thanks for both answers.

Didier, in which medium shall I conserve the isolates? Water? Lyophilized mycelium? Thank you very much!

Hi Pablo

Which Phytophthora are you working with? I know some of my colleagues from INRA Sophia Antipolis, working with P. parasitica/P. cryptogea/P. capsici, have been quite successful in keeping isolates in liquid nutrient broth, under oil, without freezing.  We have been able to keep P. infestans in autoclaved pea broth at 4 °C for about a year, but with some mortality.

Lyophilised mycelium is fine if you just want to keep dead samples for future DNA extraction, but you won't be able to restore the cultures from this type of material.

I think there are useful recipes in E. Ribeiro's Phytophthora handbook from 1975 for cultures , media, etc.... Still a good ressource!

All the best

Didier

Most of the Phytopthora isolates can be stored at  room temperature on colonised wheat leaves

Hi all

for long term storage we put agar plugs from plates in 2mL tubes with 50% Glycerol and freeze them stepwise in 12 h steps (-20, -80, liq Nitrogen) and store them in liquid Nitrogen. When freezing it immediatly in N2 we had bad experience in recovery.

Andreas,

liquid nitrogen is not available in our lab, but we have -20 and -80 °C freezers. Maybe I test -80°C conservation. How much time can you keep the isolates in nitrogen before getting them back to culture?

Didier,

I'm conserving P. capsici. The current procedure is keeping the isolates in agar plugs in sterillized water under lab conditions for about six months, but I'm searching for a more simple and efficient technique of conservation, specially when you manage a large quantity of isolates.

Thank you all!

Hi Pablo

We've had very limited success ourselves with P. infestans with freezing at either -20 or -80. It's important also to remember that freeze storage is usually meant for long term preservation - it won't be very practical to maintain a 'working collection' ( that is , isolatestyou need to manipulate very frequently  for experiments), except as a backup in case you loose some during the course of exeperimental work ( that happens!).

If you go the freezing routre, I suggest you follow the suggestion of Andreas to have a gradual process. Phytophthora has only plasma membranes (except in chlamydospores and oospores, of course), and is very sensitive to freezing. Colleagues in Scotland used to pack the samples in several layers of lab towel paper before putting them in the fridge at -20°C, to ensure a slower decrease of temperatue and a more gradual freezing process. They were quite happy with that.

Best

Didier

Thanks Didier,

good appreciation!