Yes, you know the Botrytis grows better at cool temperature, about 15°, so you need to wait iin the time

Hi Olga

I do not know if this is of assistance. As a propagator I have to reverse what we would normally do. For us botrytis usually occurs with humidity associated with cool temperatures

You are isolating fruit which you suspect is resident to the fungus. This has ethylene release issues which in turn determines how pathogens sporulate

How about isolating the complete fruiting plant by placing a clear plastic liner over the top to the ground create artificial humidity in a tent, remember that humidity can be just the breathing mechanism of the plant so water the soil only. This can be done in field or greenhouse but I deduce that this is a field experiment. Small portable garden greenhouse setups will do the same thin

This should induce population of botrytis if present without the decay mechanisms associated with fruit.

Hopefully this is some assistance, let me know as this is a new area for me, I usually hit panic buttons if I see botrytis

just get a spores from the fruit by sterilize needle by touch the spores carefully and placed it on PDA medium.

Hi Olga,

I think that you could try to put a certain amount of the fruits where you suspect the presence of the fungus in a sterilized beaker, cover with distilled sterilized water (e.g. 100 grams of fruit in a 500ml sterilized beaker and 100-150 ml of sterilized distilled water). Put the beaker on an orbital shaker and keep it  shaking  (at 150-170 rpm) for 30 minutes. Take 1 ml of the obtained suspension and serially dilute it (e.g. from D10 to D10000) with sterile distilled water containing 0.1% TritonX100  (or other surfactants). Spread 100µl of each dilution on 9cm diameter petri dish containing a conducive medium. Of course, it is highly probable that you will see a lot of other stuff, but you should also be able to see Botrytis, at least at one of the dilutions. Good luck!

thank you very much for the ideas! Karen, unfortunately I cannot create artificial beneficial conditions for Botrytis on plants and I need to sample 20-40 commercial fields for population genetics study so have to find a way to get fungus from fruit that is on the field. 

Oadi, it is what I usually do when have spores, but now the current problem is that I cannot get spores from asymptomatic fruit and this is the issue that I am struggling with.

Alessandra, this is very interesting idea! Now I changed my technique a little bit, adding surface sterilization of fruit with bleach and putting samples in moist chamber without paper towel (just on clean petri dishes) - hope it will help to decrease amount of other fungi and give Botrytis some time to grow. If it doesn't work, will try your method with shaker. Do you cut samples in half or so before shaking? I am just thinking that even if there is Botrytis it probably is still staying inside the fruit and if I should dissect the fruit to let it go to water.

Thank you very much again!

Hi Olga

As Karen tells you, Botrytis grows better at cool temperature , about 15° C

Hi Olga,

I think that to cut the fruit in half could have some advantages if you think that Botrytis is inside the fruit.. I used this technique for apples, pears and grapes, but I did not cut my samples. 

Best wishes

Alessandra

Yes, you just get a spores from the fruit of them, by sterilize needle by touch the spores carefully and placed it on PDA medium. It wll be ok!

that's right, but the problem that I am struggling with now is that I cannot get spores because of overgrowth of other fungi on samples. Trying to coll down the temperature and also want to put samples on selective media for Botrytis.

Yes, you know the Botrytis grows better at cool temperature, about 15°, so you need to wait iin the time

just put my samples in the room with 15C inside. hope it and humidity will help. thank you!

I beleive in it

Dear Olga Kozhar,

If you sure that the fungi is in Fruits, you can isolate it easily. Could you give me some knowledge about isolation procedure.

If the pathogen is infecting the fruit you can try to use DNA-based analysis to identify the DNA of the fungus using specific markers.

yes, DNA analysis might be solution but I also need to get fungus in culture to store collection that I could come back to it when needed.

The method I am using now is the following: I put fresh surface sterilized samples in moisture chambers and left them in the room with 15C and just waiting for fungus. In 5 days I can see white mycelium on some of them, not sure if it is Botrytis or no but decided to wait a little bit more.

Also want to try Botrytis selective media these days (to place samples on it).

Thank you for any fresh ideas!

Dear, Kozhar,

First of all, I was very sorry so late answer. In fact,The best solution is selective medium but I don't think your need it. Firstly, you should try standard isolation method of fungi( Agrios, 2005 ) on PDA medium.

In 1994, a long time ago, I had used paraquat to isolate Botrytis and other fungi from asymptomatic plants. Perharps this technic would be efficient to solve your problem. I join the paper, unfortunaltely in french, to this mail. Another paper, published later, described the use of a DNA probe to  identify Botrytis from one conidia. This paper was published by Katia Gindro, also joined to this mail.

Good luck for your researches

R. Pezet

you can follow the simple isolation proceduers on PDA. it is simple  

I would start with an easy selection method on Potato Dextrose Agar.

If you think Botrytis is within the fruits, surface sterilize the fruit with alcohol. Then cut small pieces of the inside of the berries and place that on a PDA plate. I would add 100ppm Penicilline and 200ppm Streptomycin to the media to prevent bacterial growth.

If you see fungal growth coming from the fruit, place some mycelium on a fresh PDA (+ Penicilline and Streptomycin) plate. Then try to infect fresh fruit with this strain to see if you have isolated a pathogenic strain of Botrytis.