I'm quantifying the proportion of cortical cells in Vaccinium roots that are colonized by ericoid mycorrhizal fungi using the 'magnified interceptions' method of McGonigle et al. 1990. The method is described for vesicular-arbuscular mycorrhizal fungi. Does anyone have advice on adapting these methods for ericoid mycorrhizal fungi?
Here's a citation to the paper:
McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., and Swan, J.A. (1990). A new method which gives an objective measure of colonization of roots by vesicular—arbuscular mycorrhizal fungi. New Phytologist 115, 495–501.
Hi Leif, what the specific problem did you meet? I will be available via emails: [email protected]
Hello Leif. I work with arbuscular micorryzal fungi and there are another way to quantify colonisation rate using Trovelout method. In this method you put the roots after trypan blue in slides and use microscope to see the colisation rate. Maybe it can help you.
I've used this method with the following mods:
Root segments, 3–6 cm long, were selected from each V. membranaceum plant, stained and mounted on microscope slides as follows: segments were first rinsed 3× in sterile dH2O and then cleared in 5 % KOH at 60 °C for 12 hr. To remove the KOH, roots were again rinsed 3× in sterile dH2O and then stained with 0.03 % Chlorazol Black E (Sigma C-1144) in 1:1 lacto-glycerol at 60 °C for 3 hr. Roots were mounted in 1:1 lacto-glycerol. Visual examination was made following the magnified intersections method (McGonigle et al. 1990), using 400× magnification and scoring within two categories (ericoid and dark septate) for a total of 100 counts per sample. (Gorzelak et al 2012).
I found it easiest to align the roots on a slide parallel to each other, and using the scale bar inside the eyepiece to determine 'intersection' every tenth bar. Start at one end of the slide, count at each intersection, scroll an entire field of view, repeat until slide is counted. I think mounting the root segments like this is randomized because you cannot tell which parts are colonized with the naked eye anyways, so it is less likely that you will bias your sample.
Good luck!
Thank you all for this help! Monica, I have carefully read your 2012 paper and it was helpful. This is a great addition. I have so far scored 5-10 of my samples and the method (slightly modified from what you used) is working. If you have any suggestions for distinguishing dark septate endophytes from everything else, I'd love to hear them.
Camila, I'll check that method out--thank you!
Hi Leif,
check out the photo from "Mycorrhizas: anatomy and cell biology" by Peterson et al (2004). Attached. 166 and 167 are dark septate; 170 and 171 are ericoid.
Dark septate are...well...dark and septate, also have thinker hyphae (167 is their microsclerotia...which will be bigger than the ericoid coils.)
Ericoid will look almost see-through, and make thinner coils that fill the cell.
hope this helps.
You could have a look at Mark Brundrett's website: http://mycorrhizas.info/
He has also written an easy to understand book: 'Working with mycorrhizas in forestry and agriculture' , which might have some pointers.
I've just uploaded our paper which reports on how we measured mycorrhizal infection of the Australian epacrid Woolsia pungens and so it should now be available for download. It contains a discussion on why we did what we did and some of the issues that we tried to deal with. I hope it will be helpful. Best wishes Anne
Monica,
Great--I'm definitely seeing both DSE and ericoid mycorrhizas in my samples, and they are consistent with those photos.
Anne, thank you. I had read one of these papers and will read the other two!
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts