Hello,
we are isolating Botrytis cinerea from fresh fruit of berries and ran into the problem with contamination of our cultures with Rhizopus. It growth pretty fast on fresh fruit we incubate to get Botrytis (all of samples are asymptomatic so we have to incubate them in the lab), and also contaminates our cultures. We use all required sterile techniques and everything but still dealing with Rhizopus.
Could anybody suggest how to get rid of Rhizopus - prevent contamination of cultures and clean already contaminated ones? Maybe some specific fungicide with high selectivity to Rhizopus but not to Botrytis?
Thank you very much for any suggestions
Olga
Dear Olga Kozhar
The need to sterilize laboratory accurately
With Fumigation lab
Observe the berries under stereo microscope where pure colonies of Botrytis present on the surfaces (without any Rhizopus contamination in large extent). For isolation, place a drop of water on Petri plate containing MEA medium (prepare a thin layer as much as possible then only you can observe under microscope), then using a sterile syringe (dip a tip into sterile water) slightly touch the conidial head of Botrytis and transfer into plate where you dropped the water. After placing lid, observe the plate upside down under compound microscope using 4x/10x objectives for the presence of spores. After 24 hrs, observe the plate again for germination of spores, then immediately transfer the germinating spore into new petri plate containing MEA/PDA medium.
This is a simple method and you need not to use laminar flow chamber for isolation.
For purification of contaminated cultures, do serial dilution and transfer 100 µl onto MEA medium. Observe the plates for the appearance of colonies. The fast growing colonies can be removed using sterile scalpel.
I am in general agreement with what Senthilarasu Gunasekraran writes. I would point out that when working with either Rhizopus or Bortrytis it is advisable not to work under an operating laminar flow hood as anything downwind will be contaminated by air- liberated spores.
Dear Ogla Kozhar,
I prefer to use the any selective medium in isolation such as that (Edwards and Seddon, 2001)
http://onlinelibrary.wiley.com/doi/10.1046/j.1472-765x.2001.00857.x/abstract
one the way, you need sterilize your laboratory by fumigation and you can used cabrio fungicide.
Best wishes,
Mahmoud
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