I'm trying to make a collection of slides of the 4 Sonchus species found here in the U.K. Many of you will have seen my earlier posts (in fact right from my very first adventures back in 2015...) often featuring Sonchus species, namely S.asper (prickly sow-thistle) and S.oleraceus (smooth sow-thistle). The remaining 2 (until now...) being S.arvensis (perennial milk-weed) and S.palustris (marsh milk-weed).
I say until now as recently I discovered a small number of Sonchus.arvensis growing on the sides of a bay near where I live! Gathering a specimen and placing straight into formol-acetic-alcohol 'fixative' I disappeared into the murky-depths of my tiny 'lab' at home....
Here's an image depicting Sonchus.arvensis from wiki-commons, I didn't think to take an image before beginning to process the tissue!
- ws_500W_small_Sonchus_arvensis.jpg (45.64 KiB) Viewed 13490 times
This is what the infection looked like in preservative....
- ws fungus labelled 1.jpg (85.1 KiB) Viewed 13490 times
- ws fungus labelled 2.jpg (40.75 KiB) Viewed 13490 times
So, what do the sections look-like stained & mounted permanently?
I'm pretty pleased with the slides, which I also used as 'testers' for staining. I've been practicing and trying to perfect the combination of Alcian-blue and Safranin as a bi-stain, and then the addition of Orange-F to make a tri-stain. This turned out to be easier said than done!
Here are some images of early results, first-up a section taken (TS) across a leaf-node - the point on a stem at which a leaf is attached, and the axil between the leaf and stem which invariably contains an 'axial-branch' which will be a stem or flower etc, but not another leaf. This unit of growth - the leaf-node with axial bud/branch is the way flowering plants develop.
Here's an idea of where the section was made,
A section through this plane shows signs of the fungal attack on both the stem and leaf epidermis,
TS through infected leaf-blade shows fungus breaking through epidermis,
These images are tr-stained with Alcian-blue, Safranin and Orange-G,
The leaf-blade,
Stem cross-section
back soon
This is the edge of an infected leaf, showing the structures of the fungus bursting through the epidrmis of the unfortunate Sonchus, stained with Alcian-blue and Safranin combined bi-stain, 8µ sections.
I also sectioned thinner - at 5µ, and stained with the tri-stain, Alcian-blue + Safranin + Orange-G in an attempt to bring-out more details from the fungal tissue. The idea with the addition of the Orange-G is that the Orange-G will displace selectively some of the Safranin which may have over-stained some structures and reveal a more subtle and detailed staining result. It certainly did seem to work once I managed to get the proportions, concentration and staining-time factors optimised....
This is a 5µ section through fungal structures with the tri-stain applied, again bursting through the lower-epidermis of a leaf,
and a stem,
This fungus I have since identified, with advice from the UK fungus forum's helpful folk, as a 'rust-fungus', one of only two types specific to Sonchus species, the Miyagia.pseuosphaeria. These 'micro-fungi' can have up to 5 different spore-types! I've still to identify exactly which types these are but there are certainly more than one present - and in some later images there appear to be fungal-hyphae infiltrating the cells also, a result of the germination of one of the spore-types....
Closer-in more structure is revealed, aided by the slightly less opaque stain from Orange-G that has displaced some of the rather too dark Safranin of the earlier images,
and
The two-stain does give a lot of detail, as this next image shows, but I still think the tri-stain edges it with this very fine tissue,
The Alcian-blue and Safranin does give a beautifully colourful slide with great contrast,
I like this image, where the fungus is 'up against' the mid-vein of the leaf. The tri-stain here hasn't worked as intended, although I like the result. Here I've over-stained with the Orange-G, which has almost completely removed the Safranin (the order of staining I use is, Alcian-blue, then Safranin then Orange-G)... An adjustment soon put this right however (after about another 10 hours of trial and error
).
The 'greenish' shades of this tr-stain are the result of interaction between the Orange-G and the Alcian-blue - again this may be altered by varying the staining-strengths of the two stains - so many variations and combinations! This will also change on a per-plant basis also to a larger or smaller extent - staining is indeed a dark-art!
Now, I mentioned above the presence of hyphae.... These result from the germination (upon the Sonchus epidermis) of the spores produced by one of the other stages also present on the plant - this fungus is said to be 'autoaecious' in that it lives it's cimplete life-cycle on the one host plant. Some fungi go through some stages on one host then complete their life-cycle on another, secondary host.
Here's a section of stem where the germinating spore (a 'teliospore' I think) has produced a hypha (the tube) that finds it's way by responding to ridges on the host's epidermis or cuticle until it literally 'find an opening' - this opening will be a stomate-pore or perhaps a wound.... In this section the path of infiltration can be seen. The hypha actually appears to be heading for the vascular-bundle of the plant - probably for the phloem as this is where the sugars or 'foods' produced by the plant are transported from the leaves to the plant'e tissues....
Close-in of the mangled tissue that used to be a stomate and sub-stomatal chamber - full now with fungus I think,
This image I like - it shows clearly how the hypha (stained red) makes it's way through the tissue between cells, the so-called apoplastic pathway rather than through cells - the so-called symplastic pathway...
I think these are hyphae that are within the mesophyll (immediately below the leaf's epidermis) of an infected leaf - the Orange-G has stained the hypha/e while the Safranin has picked-out the nuclei in red....
These images show just how invasive and therefore devastating this rust-fungus (and presumably others) can be!
and,
Here's another entry-point - the stomatal pore is mid-left on the epidermis - the hyphae are quite rampant!
and here the open stomate is visible 'properly',
Spore-producing structures have matured behind a stomate here, but haven't (yet...) ruptured the epidermis to release the 'next stage' spores onto the suffering Sonchus,
Finally for now (until I can take some more images from the still-drying 5µ slides) I think I have some hyphae outside the plant's body, creeping along the epidermis in the first image,
and along the edge of a wounded-edge (maybe result of the fungus' invasion or a wound from something else) that looks like a 'bit has been taken out' of the plant.... The red is the edge, the orange is the hypha,
Anyway, apologies for the long post - I got a little carried away with this interesting fungus!
I'll post more when I've some more images of interest, hope you find these interesting in the man-time...
John B.
) here in the U.K. - I find them simply fascinating, and now I make my own slides I'm in a 'Botanical Heaven'!! 