i currently isolating different fungi from the soil by soil-plate and serial dilution method. i am interesting to know that is there method to identify the mycorrhizal or saprophitic fungi from these soil-isolated fungi. please give your valuable suggestions in this regard. Thanks in advance
Dear M Z Haider
First of all you have to know that mycorrhizal fingi are obligate biotroph.................that means they can not be culture without the presence of host root. recently, interests were given on cultivating on prepared media...........some researcher were working in this directions in India. but............... not well established.. So, most or all of your isolated fungi will not be mycorrhizal at all.
I agree with A Singha although this is mostly applied to VAM fungi. Hoever many basidiomycete ectomycorrizal fungi can be grown in axenic cultures. So it is posible that some of your soil isolates could be some of these. They don't produce spores and many can have clamps. For identification I prefer the molecular way.
Best wishes
I agree with both previous contributors - it is almost impossible to grow VAM fungi on agar plates. Some ectomycorrhizal fungi can grow on agar plates, but the majority of isolated fungi from serial dilution methods are saprophytic fungi. The best way is to identify all isolates by morphological or molecular methods and find their role in the environment by literature search.
Best wishes.
I also agree with J. Jirout. The majority of isolated fungi from serial dilution methods are saprophytic fungi but I recommend to make a thoroughly observation of the fungal colonies that appeared in the plates looking for those that do not sporulate after 1-2 weeks (please include Triton-X-100 in the agar medium to restrict the growth of colonies) and pay attention to hyphae because some basidiomycetes have clamps. These colonies cannot be identified by morphology so you must go for molecular identification. There are some media that promote the isolation on basidiomycetes and also there are some tricks to be used if you are looking for those that can degrade aromatic compounds. Good luck!
@ dear Atul Singha . yes you are right that in most of the cases the soil isolated fungi are saprophytic but we can grow the mycorrhizal fungi in lab except VAM (obligate biotrophic).
@dear Marcel Gutiérrez-Correa. yes you are right that now a days we have to trust only molecular data for identification. but i think we first required some kind of initial screening. What you suggest for molecular identification? sequencing with 18S or 28S? or there some other marker?
i could not understand the role of triton-X-100 in identification. Could you please elaborate it?
is there any importance of C/N ratios in the growth medium to favor the growth of some kind of fungi over the others? thanks for your time and nice suggestion
Dear M. haider,
Hope you are doing well,
There are many differences between VAM fungi and Saprobs! As you know, VAM fungi are oblige and produce Chlamydospore-like spores, but soprobs are not VAM. For saprobs, you can use from Atlas of soil Ascomycetes, This book has key, description and illostration.
I think this can help you about some isolates of saprobs that you have.
All the best,
Amir
For VA mycorrhizae & Ascomycetes microscopy is the simplest but of course you need to know their characteristic structures & classification, for which you need to have keys to their ID. As for Ectomycorrhizae & Basidiomycetes the molecular techniqes such as ITS PCR or 18S-DNA can be used for the ID.
For long periods of incubation, the confluent growth of fungal colonies a problem for their isolation and study that is why the use of triton-x-100, which is a nonionic detergent, is useful because it reduces the diameter of the colonies. Basidiomycetes may have a slow grow and their colonies can be overgrown by ascomycetes and, worst, by zygomycetes. (I assuming a general objective for isolating basidiomycetes from soils but if you have an specific objective you may look for other methods).
Once you have selected colonies of slow growth, without spores and better with clamps you can isolate them for further studies; they may probably be basidiomycetes. For identification you should extract DNA and perform PCR using ITS primers; after purification of PCR products (check for the size according to the expected in silico analysis) send them for sequencing and do the appropriate bioinformatics analysis.
Hi Muhammad,
get a sequence of the rDNA-ITs region. blast against UNITE (http://unite.ut.ee) - there are information there about the current state of knowledge for the mycorrhizal status of each species hypothesis.
however All fungi isolated from soils have the potential to function in symbiosis with plants, depending on plant species and conditions. we still have very limited knowledge of what is or not capable of functioning as mycorrhizal symbionts.
You could set up bioassays if you have access to sterile culture facilities. but it takes time and effort - so it depends how important it is for you to find out.
cheers
thanks all for your nice suggestions.
it is really very interesting that now fungal identification is shifting towards the genetic base identification. but i have still a question that is it sufficient that a single DNA barcode marker like 18S/28S can be used for fungal identification or it required more than one DNA barcode marker? In my opinion, the species confirmation through blastN results is also very difficult because a lot of similar sequences are also registered with different fungal names /and strains on NCBI public data base. is there any other website like unite (mycorrhizal) for all the fungal sequences?
Thanks again for your time
UNITE (unite.ut.ee) is for all groups of fungi, not only mycorrhizal. It focuses on ITS sequences, the formal fungal barcode. In my experience it works well for most groups of fungi.
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