They are most likely an oomycete, as you suggest, but it is unlikely you will be able to identify them. They are not arbuscular mycorrhizal structures. I suspect your hanging roots are rather wet - lovely conditions for Pythium, etc, but not so good for AMF. By the way, trypan blue is dangerous. Change either to ink or methyl blue if you can.

You can try to isolate Pythium spp. on a selective medium CMA-PARP

see Le Floch G, et al 2007 Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA macroarray and real-time PCR. FEMS Microbiology Ecology. 61(2):317-326.

I have used this staining procedure for Pythium spp on tomato roots you can see the result on attached file.

With the picture

sorry

this is not my specialty but they might be arbuscules in the roots. I had seen a similar structures in an article about "Tropaeolum majus" stained with Trypan Blue. (An overview of methods for the detection and observation of arbuscular mycorrhizal fungi in roots).

Hi

Fig. 1 Stained root of Hieracium pilosella, colonized by Rhizophagus intraradices (a) with arbuscules (b) and vesicles (c).

http://web.biologie.uni-bielefeld.de/systemoekologie/index.php/de/research-projects/mycorrhiza

Are you sure it is R. intraradices? Most of the things with this name are something else. The vesicles look like spores. I reiterate, Trypan blue is a hazardous substance, so may be better to think of some other stain.

This stupid system only allows one attachment at a time. Here's another on ink staining.

Another on how to keep arbuscular mycorrhizal fungal cultures free of other AMF contaminants.

Hi Peter,

I would recommend to check the alignment for Koehler illumination of your microscope.

For your specific question, Christopher Walker gave you excellent advice, both on the diagnosis and on the staining. Stay away from trypan blue, if you can. You can also check:

Vierhelig et al., Appl Environ Microbiol. 1998 December; 64(12): 5004–5007.

Ink and Vinegar, a Simple Staining Technique for Arbuscular-Mycorrhizal Fungi

Dear Peter

One of the most important things in Biology is good observation, the big WHY behind the invention of different microscopes with higher resolutions and magnifications. You surely live in a Country that You can repeat your observations even experiments. Then, would You please take more photos each time focusing well on different organs You see. Maybe You have both symbiotic and parasitic infections, and perhaps only with an oomycetous organism. With each of these cases You will get Pythium on an specific medium, and You will get only endomycorrhizal AMF when You have only them in Your sample.

surely, the picture shows the infection by oomycetous fungus. Kindly confirm after culturing on PDA medium.

I agree to consider that these structures are not mycorrhizal fungi, it would be desirable to consider use other dyes , this look like "oomycetes structures"

I agree with Christopher Walker. They are not arbuscular mycorrhizal structures. Also only on the basis of staining one can not be sure of the fungus. One has to now confirm by molecular sequencing techniques for the identification for which you have to isolate and get the pure culture of the suspected fungus. If you have fungal monograph then you can identify the fungus but molecular technique is the better choice for the identification.

Dennis, these are not "sections", but root pieces squeezed between the slide and the cover glass.

I stick to the point that the microscope is not well set.

Dear All,

Many thanks for your replies!

Christopher, I do agree with you, we thought that these structures were most likely oomycetes, however this is the first time I have observed oomycetes stained in roots and I am grateful for the confirmation from yourself and others. In regards to the dangers of trypan blue, I am aware that it is carcinogenic, however I am experienced in using it safely, an we only work with it in a high quality fume hood, with full protective equipment and fully risk assessed. In the future I will certainly consider using other stains, and I am grateful for the references you have sent, however at the moment spare time is very hard to come by with having such a large experiment under way along with other responsibilities, and until I can find time to test other stains, we will have to stick with trypan blue for the time being.

For those who have suggested culturing or using molecular techniques to identify...I would certainly look to do this in an ideal world, however the purpose of this test was simply a test stain to identify the presence / absence of AM fungi as we were using the plants to set up an experiment with various AMF communities (and as such plants without AM colonisation were required). The oomycetes observed were present across all of our plants, and as such their effects, weather positive or negative, should remain constant across all treatments. The plants all seem perfectly healthy at the moment, so it does not seem to be having a deleterious effect on them.

For those who advised that more sampling should be done, we did sample a representative sample of all plants, and more pictures were taken, however I only included one in the first instance as it seemed to be fairly representative of what I was observing. I will, however, add more pictures for reference. Just to note, hyphae were not observed throughout the samples.

Thanks again everyone for your help, I really appreciate it!

Peter

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Trypan blue in lactophenol was used succefully for staining arbuscular mycorrhizal fungi in roots to show arbuscular, vesicles and internal hyphae. I think we can used this stain in oomycetes and hyphomycetes to stain the spores but very rarly used.

Guido, any advice on how the microscope setup can be improved would be appreciated. I do agree the alignment for Koehler illumination seems to be off and steps will be taken to check and correct this. Any other advice?

Thanks again all!

Hallo, I do agree with Oomycetes, most probably some resting spores in root structures, no flagella, they are very minute. Or, might be, that some plant protoplast was not destroyes inough, and there are some artefacts left.

But see this ancient article: http://www.cabdirect.org/abstracts/19711101080.html;jsessionid=53DB00084D164253D157A65A30089A81

Peter,

here are two tutorials on the alignment for Koehler illumination:

http://zeiss-campus.magnet.fsu.edu/articles/basics/kohler.html

http://www.microscopyu.com/tutorials/java/kohler/

It is rather easy and you can do it own your own. Usually, the smallest objective you can use to set the alignment is 10x: if you have a 3.2x or a 5x, most likely you won't be able to do it (but this may depend on the brand of the microscope).

It is important to modify the settings with each objectives *especially when you want to take pictures* (for ordinary observation you do not need to change the settings any time you change magnification).

Apart from this, the other step is to have as good staining as possible. So, have a nice clearing of the roots (no problems in this, as far as I could see from your pictures), stain them with the protocol of choice and wash them thoroughly. That should be enough.

In the animal, field Trypan blue has been used to visual cell death,

I have attached below an abstract where they have used trypan blue for this. I am susciptious that the structures are fungi, or oomycetes but I am not specialised with oomycetes. Looking at your pictures it looks like whole cells are stained, could it be that they are undergoing cell death? Only a suggesion, I am not sure.

Trypan Blue Dye Enters Viable Cells Incubated with the Pore-Forming Toxin HlyII of Bacillus cereus

X Maud Ngo-Camus, Affiliation: INRA, Unité MICALIS, UMR 1319, Guyancourt, France

X Nalini Ramarao mail * E-mail: [email protected]

Affiliation: INRA, Unité MICALIS, UMR 1319, Guyancourt, France

X Published: September 06, 2011

•DOI: 10.1371/journal.pone.0022876

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.

I am with dear Laurence, as the intracellular objects seem as artifacts resulted from the penetration of trypan blue color into the cells disrupted during sectioning process. You see no hyphae around these objects mistakable with vesicles of AM fungi. But You see also no arbuscules (dendroid haustoria of AM fungi) required for the absorption of the nutrients from the plant partner if there is an endomycorrhizal relationship formed, what seems absent based on these photos.

I agree with those that think in artifacts due to penetration of the staining solution into root cells.

I agree with all the suggestions and commentaries. If there is an AMF infection you must observe hifa, mycelium and arbuscles also. And the vesicles look different than the estructures that you showed in the foto.

Regards

Gladys

I agree with Laurence and others. Whole cells are being stained suggesting cell death. I do not see any clear haustorium-like structure indicating there is an oomycete in your sample. Haustoria do not usually occupy a large portion of the plant cell, except for those of very few rust-causing fungi.

I agree with the comments. These 'round graded' structures are Oomycetes. They can occur in many different plant species. I found them in herbaceous, grass but also woody species from different ecosystems ranging from lowland meadow, garden, forest up to alpine tundra.

Light blue small structures are nuclei. Their presence (but also 'brownish' color within the cells) indicate too short boiling with KOH. You could try to boil them longer in KOH and use 3% solution of H2O2 to remove them after maceration in KOH. This need a lot of training but at the end you will fit your procedure to the different roots or roots of the different age - this is also important. I would recommend you also to try ink staining, but then KOH step should be done perfectly in order to visualize fungal structures. Good luck.

Oomycetes do have coenocytic hyphae and if something like Pythium invades plants it causes cell wall disruption and soft rot. Here in this photo and based on what is observed, we can not observe aseptate hyphae, oospores are produced in oogonia on oogoniphores and as the result of fertilization by antheridia or perhaps without antheridia in some cases. The provided photo does not indicate these requirements for the existence of oomycetes.

https://www.researchgate.net/publication/216899187_Diversity_of_arbuscular_mycorrhizal_symbiosis_in_plants?ev=prf_pub

Article Diversity of arbuscular mycorrhizal symbiosis in plants

Hello,

this seems to me like mostly empty sporangia of the chytridiomycete Olpidium brassicae. This an obligate biotrophic fungus forming zoospores that can infect rhizodermal cells. Characteristically is the holocarpia - which means the whole biomass is after the Initial amoeboid development transferred completely into sporangia. The small pinpoints are the channels formed to release the zoospores to the root surface.

Later on characteristic thick walled permanent spores can be formed. In the lower part of the picture the sporangia seem to contain still zoospores - but these are not really in focus.

Olpidium species have a very broad host range and can be found abundantly under field conditions in large spectrum of host roots - especially under high moisture and runing water around the roots.

Olpidium brassicae is causing damping off of seedlings at crucifers, at cereals and solanaceae the damage is quite low. But due to the life cycle the fungus can trasmit plant viruses.

Chemical control is quite difficult - almost no fungicides are available against this pathogen to my knowledge.

To avoid further spread I would suggest to start AM fungal cultures again from single spores or selected "clean" root pieces with AM. Sporangia and resting spores are also visible without staining in living roots.

The period ot water application to the root should be reduced to shorter intervals.

I would further recommend to make use of inorganic carriers for inoculum production of AM fungi - see publication in my records (Dehne & Backhaus) - good luck!

Hello again,

additionally I want to give the papers - sorry these are in German - from 1986 and 1989. Additionally find enclosed a patent available at "espacenet" for download with an english description of inoculum production AMF at inorganic carriers (Baltruschat & Dehne, 1985).

AM fungi tend to grow into ecological niches - and carriers can provide cracks and wholes and they form chlamydospores there. Description in the paper of Grunewaldt-Stocker & Dehne, 1989. So the inoculum can easily be separated from plant roots, dried and stored for years with high effectivity.

Additionally you may rinse this material with 30 % alcohol/water for 2-3 minutes and rinse intensively with plain water to eliminate the alcohol. This will eliminate all zoospores efficiently - those from Olpidium as well as Pythium and Phytophthora species that may contaminants of roots.

The carrier material which is most suitable to our experience is broken expanded clay which is used for hydroponic culture systems - e.g. hydro green, broken with fraction 4-8 mm. Can be ordered by various sources via internet. But it should be a material releasing only low concentrations of ions interfering with plant growth. We have best experiences with Lecaton.

If you start with clean inoculum and reduce water availabity Olpidium should be no further problem. We found an interaction of AMF with Olpidium - but it is a question who comes first ! And also a question of inoculum density as well as environmental conditions.

In a survey of you may be lead to David Sylvia - he has done a lot of inoculum production of AMF in aeroponics. This may help you too ... hopefully!

Best wishes

Sorrry but only 1 pdf possible - here come the announced ones ...

now the patent info - look for espacenet research - there is the complete download available (Baltruschat & Dehne, 1985 - Company: Chemische Werke Hüls)

With season's greetings all the best for 2014 - Heinz-W. Dehne

sorry, I forgot to mention: After holocarpia process is finished, the particular host cell is somewhat "exhausted" - not getting necrotic, but due to formation of bound residues of assimilation products as well as impaired membrane function stains stay within these cells, typically only in single contaminated / infected cells - regardless whether ink, trypanblue oder others. For observing vital functions I like most to get accustomed to unstained microscopy and living cells. Again all the best !

It could also be a member of cryptomycota. Please try to extract DNA and do the phylogenetic analysis.

With chytridiomycetous zoospores or of cryptomycota planospores, they encyst on root surface and the short germ tube infects the most superficial layer of root cells. Here in this photo if You have taken them from inner sections, then it would be impossible to see holocapic forms of such fungi in such deeper parts. They can be found in deeper parts if they disrupt plant tissues that would be observable in this case at least microscopically.

As I understood this is a preparation according to identify arbuscular mycorrhizal fungi by conventional clearing and staining procedure. Therefore the structures are found in rhizodermal cells only. The 3-dimensional presentation kann also be identified, because only the cells in the middle are in focus and cells on top and bottom of photograph are increasingly out of Focus.

They are not arbuscular mycorrhizal structures.

Better do plate culture of the fungus.

Culture plates will not be possible Olpidium species are obligate biotrophic plant pathogens, sorry - but definitively this is no arbuscular mycorrhizal fungus !

Yes, as I said a while ago, there is no doubt in my mind these are not AMF. They (AMF) also are obligate symbionts, so you would not have any success with plate cultures (except for a few seemingly atypical spp. that can be cultured in root organ culture (ROC). The images are in fact quite sufficiently good to know that these are not glomeromycotan fungi.

Definitely not arbusuclar mycorrhizal fungi. I agree with Babak Pakdaman Sardrood. They are the cryptomycota aplanospores

Trypan blue could be used to stain all fungal structures like spores, vesicles, arbuscular anh hyphae. In order to show all the structures easily , the tryban blue stain is the best stain for this.

No vam

I agree with all comments made before - again this is definitively no AM fungal structure! The infected cells are clearly rhizodermal cells from a total preparation of root peaces - supposedly by clearing and staining procedure (as described by David Hayman and others). Although out of focus in the lower part of the picture a complete sporangium as well as characteristic early resting spore. Restart of am root propagation from single spores is recommended.

Why do so many people ignore the fact that trypan blue is a hazardous substance? With regard to Gamal Abdel-Fattah's comment, trypan blue is not the best stain (there is no such thing, as some AMF stain better with different stains). As a general stain, I use Quink blue ink (this presumably must be safe as it is marketed to the general public for use in pens) or methyl blue (which seems to be safe according to safety data sheets). In case you think that these stains are not so good as trypan blue, here is a methyl-blue stained mycorrhiza and another stained with Quinik (difference in intensity is a species matter, not to do with the type of stain). NOTE: it is not clear if both have attached. I added the methyl blue first and the ink second, but the first image filename disappeared when I uploaded the second one ...

Hmmmm. So, as I thought, only the second one loaded. Here is the methyl blue one again.

Chlorazol black E (CBE) in lactoglycerol, is used as staining,0.03% w/v is best in most cases, but it is best to try a range of concentrations (0.1%, 0.03%, 0.01%) when using this procedure with a new type of roots, or when using a new source of dye.

Acid fuchsin and cotton blue can also be used to stain fungi in roots, but destain rapidly and produce low-contrast images (Brundrett et al. 1984)

Other is 5% ink diluted in vinegar (5% acetic acid). Staining with black writing inks (Shaeffer Jet Black; Cross Black; Pelikan Black) and some blue inks (Pelikan Blue) give good staining results.

BUT Chlorazol Black is very hazardous. Why use it when a safer alternative is available? There isn't a 'best' stain because it depends on the host species and the fungal species concerned (I have used Chlorazol too). Similarly, I have tried numerous black and blue inks, and they don't always work. Some glomeromycotan species don't seem to stain with anything, and in one case, Claroideoglomus claroideum stained perfectly well when with corn, but not at all with apple. So, the best thing, I suggest, is start with a non-hazardous substance and only if necessary move on to a more hazardous one (ensuring all safety precautions are taken). Paraglomus spp. seem to be very difficult to stain, and these can be visualised in cleared roots by using Differential Interference Microscopy (Nomarski).

yes, I agree not to use hazardous dyes. Of course there are other alternatives such as those mentioned by Walker. If you have a good microscope, you can also use the DIC, and also can be evidenced by autofluorescence fungal structures, especially when they come from contaminated soils by Al and others heavy metals, this, what I have observed in my laboratory.

If some researchers could read this discussion!

I agree with Dr. Chris Walker. These don't look like mycorrhizal fungi. Staining with Trypan Blue is not a confirmation for AMF.