# Need a soil expert!



## JoshP12 (29 May 2022)

What are all these crazy colours!?







For reference:


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## Simon Cole (30 May 2022)

Epic photograph!
Where plant roots interact with soil and microbes we call the rhizosphere.
The "blue and green" patches appear to be abiotic microbial colonies forming in two separate soil horizons and it looks like they are consuming organic molecules from the soil itself.
The long vertical root in your photograph is a bit more interesting. It has a reasonably well-defined root microbiome and three associated microbial colonies are easily visible: The top root section is more mature and has a "black" ectotrophic sheath. This is surrounded by a "white" microbial horizon that is fairly passive in association. Further down the root there is an "orange" colony that starts on the epidermis of the root. It widens into a horizon offset from younger root tissue, so it fairly facultative in association and displays evidence that the root zone has chemical inhibition.

Generally speaking, roots are surprising capable of modifying their root microbiome. If we look at the zone displaying inhibition of the "orange" microbial community, then you can see from the diagram below that this could be due to chemical root exudates like hormones (ethylene, salicylic acid etc.) or metabolites (phytoalexins, triterpenes, benzoxazinoids etc.) that are limiting it's spread, but equally, the roots could be stimulating beneficial microbes to either defend-against or outcompete the "orange" colony. _Trichoderma harzianum _is a classic example of an endotrophic fungi that is in a symbiotic relationship to defend plants from undesirable and pathogenic microbes. Trichoderma will often grow within the plant from the point when it starts life as a seed, migrating downwards into the root and providing a chemical (antimicrobial) barrier that stops the infestation of other microorganisms and pathogens. Interestingly, Trichoderma is equally capable of preventing mildew in leaves, and we can therefore say that this symbiont provides systemic plant defence. The thing that <always strikes me> when we get posts talking about "melting" in-vitro plants is that people are overlooking the most obvious causes; that being - immunity to defend against opportunistic pathogens. Personally I would like to see more products that establish aquatic root microbiomes, especially biological inoculants (symbionts) that could actually prevent Bucephalandra melt.



If you think about plants, then they are incredibly careful curators of their own root microbiomes. They know exactly what they want, who they want to achieve it, and how to go about getting the results.


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## JoshP12 (30 May 2022)

Simon Cole said:


> Epic photograph!


😃😃


Simon Cole said:


> Where plant roots interact with soil and microbes we call the rhizosphere.


Up until this point, the rhizosphere has been a cartoony diagram and a abstract concept with immense power —- When I took the photo I had a hunch it was the rhizosphere but had never “seen one”. 


Simon Cole said:


> The "blue and green" patches appear to be abiotic microbial colonies forming in two separate soil horizons and looks like they are consuming organic molecules from the soil itself.
> The long vertical root in your photograph is a bit more interesting. It has a reasonably well-defined root microbiome and three associated microbial colonies are easily visible: The top root section is more mature and has a "black" ectotrophic sheath. This is surrounded by a "white" microbial horizon that is fairly passive in association. Further down the root there is an "orange" colony that starts on the epidermis of the root. It widens into a horizon offset from younger root tissue, so it fairly facultative in association and displays evidence for a root zone with chemical inhibition.


Stages of microbial growth ?! Is possible that if. I had paid more attention, the black and white group could have “been similar in visual to the orange section”. 


Simon Cole said:


> Generally speaking, roots are surprising capable of modifying their root microbiome. If we look at the zone displaying inhibition of the "orange" microbial community, then you can see from the diagram below that this could be due to chemical root exudates like hormones (ethylene, salicylic acid etc.) or metabolites (phytoalexins, triterpenes, benzoxazinoids etc.) that are limiting it's spread, but equally, the roots could be stimulating beneficial microbes to either defend-against or outcompete the "orange" colony. _Trichoderma harzianum _is a classic example of an endotrophic fungi that defends plants from undesirable and pathogenic microbes. Trichoderma will often grow within the plant from the point when it starts life as a seed, migrating downwards into the root and providing a chemical (antimicrobial) barrier that stops the infestation of other microorganisms and pathogens. Interestingly, Trichoderma is equally capable of preventing mildew in leaves, and we can therefore say that this symbiont provides systemic plant defence.


😍


Simon Cole said:


> The thing that <always strikes me> when we get posts talking about "melting" in-vitro plants is that people are overlooking the most obvious causes; that being - immunity to defend against opportunistic pathogens. Personally I would like to see more products that establish aquatic root microbiomes, especially biological inoculants (symbionts) that could actually prevent Bucephalandra melt.


Is Bacter 100 by ADA - the substrate additive - a “potential way that ADA attempted to do this”?


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## Simon Cole (30 May 2022)

Simon Cole said:


> Personally I would like to see more products that establish aquatic root microbiomes, especially biological inoculants (symbionts) that could actually prevent Bucephalandra melt.





JoshP12 said:


> Is Bacter 100 by ADA - the substrate additive - a “potential way that ADA attempted to do this”?


I suspect not. _Trichoderma harzianum_ and _Bacillus subtilis_ are arguably the two most common biological control agents (BCAs) used in terrestrial horticulture/agriculture. They are both symbionts that function to protect plants from infection. They are endotrophic microbes that migrate within plant tissue or sporulate in the air. If ADA had similar strains of BCAs in Bacter-100, they would want to ensure that they were suited to submersion. Plant tissue is relatively well oxygenated and often has air pockets, so many of these spores should be fine once they have inoculated themselves within aquarium plants. But this assumes that they can both survive and penetrate into plants while they are submerged in the aquarium soil. A better option would be to foliar spray these BCAs directly onto plant leaves, or introduce during the in-vitro stage of cultivation, or even better, use them during an aquarium dry-start. That should all work well, but might not be absolutely essential. It's just that lots of things could consume or damage these BCAs before they even get the chance to inoculate aquarium plants, so I would prefer to take the short-cut.



 Shown left: Trichoderma attacking a plant pathogen.

I also cannot see how ADA ended up with nearly 100 microbes. There would be a high degree of inter-microbial competition, and getting a compatible and effective multi-microbial BCA would be increasingly difficult. Storage would also be a challenge (I tend to keep mine in a fridge). Generally you keep inoculants separate, relatively sterile, and inactivated; certainly knowing whether they are compatible in the first place. I can think of up to 16 BCAs that have been used together, but nowhere near 100 (and in any event you probably only require one or two; _Trichoderma T22 _ being one of the best).
Some of ADA's microbes might be mycorrhizal (fungi capable of forming a mutualistic association with plants), but it would take a bit more to demonstrate that they were effective BCAs...
It's a bit like substituting somebody's grandmother for goalkeeper - still on the same side, but not quite in the same league of defence.

ADA have also not clearly identified what is included. At least with something like Great White Premium Mycorrhizae you know the ingredients.
I am not sure whether ADA intend it to be a mycorrhizal (fungal) product. They call it a "substrate bacteria" so it is hard to determine [symbiotic] benefits or potential negatives to aquarium plants. Moist garden soil might have similar properties to ADA 100. Whereas with Great White, you know that it contains the following (and this is quantified) - it would be my first choice:

_Endomycorrhiza:_​_Glomus aggregatum – 83 props per gram
 Glomus intraradices – 83 props per gram
 Glomus mosseae – 83 props per gram
 Glomus etunicatum – 83 props per gram
 Glomus clarum – 11 props per gram
 Glomus monosporum – 11 props per gram
 Paraglomus brazilianum – 11 props per gram
 Glomus deserticola – 11 props per gram
 Gigaspora margarita – 11 props per gram__Ectomycorrhiza:
 Pisolithus tinctorious – 187,875 propagules per gram
 Rhizopogon luteolus – 5,219 props per gram
 Rhizopogon fulvigleba – 5,219 props per gram
 Rhizopogon villosullus – 5,219 props per gram
 Rhizopogon amylopogon – 5,219 props per gram
 Scleroderma citrinum – 5,219 props per gram
 Scleroderma cepa – 5,219 props per gram__Bacteria:
 Azotobacter chroococcum – 525,000 CFU’s per gram
 Bacillus subtilis – 525,000 CFU’s per gram
 Bacillus licheniformis – 525,000 CFU’s per gram
 Bacillus azotoformans – 525,000 CFU’s per gram
 Bacillus megaterium – 525,000 CFU’s per gram
 Bacillus coagulans – 525,000 CFU’s per gram
 Bacillus pumilus – 525,000 CFU’s per gram
 Bacillus amyloliquefaciens – 525,000 CFU’s per gram
 Paenibacillus durum – 525,000 CFU’s per gram
 Paenibacillus polymyxa – 525,000 CFU’s per gram
 Saccharomyces cerevisiae – 525,000 CFU’s per gram
 Pseudomonas aureofaciens – 525,000 CFU’s per gram
 Pseudomonas fluorescens – 525,000 CFU’s per gram__Fungi:
 Trichoderma koningii-187,875 CFU’s per gram
 Trichoderma harzianum-125,250 CFU’s per gram_
​People can also grow their own mycorrhizae so that they know their inoculant is activated using the autoclaved "rice bag" technique (something cheaper based around the "PF-Tek" method might also work). If I was growing In-vitro plants, then I would find it hard not to want to add some BCAs to my agar jelly. You could even potentially activate an aquarium soil, by inoculating it and incubating it in the same way. Going a bit off-piste, this would be fun because we could potentially see mushroom growing aquariums, which would make an interesting IAPLC entry. Obviously they would need to manage fertiliser to eliminate potassium sorbate and reduce sulphate levels, but yes, people might be able to grow certain aquatic mushrooms if they inoculated and incubated their aquarium soils first.



JoshP12 said:


> Stages of microbial growth ?! Is possible that if. I had paid more attention, the black and white group could have “been similar in visual to the orange section”.


The "black" group looks like an ectotrophic microbial colony to me (possibly fungal), and probably living on older plant roots that are not exuding growth inhibitors. It could be obtaining nutrients from dead cells on the surface of the root, penetrating and infecting the root to draw nutrients, or simply colonising the surface opportunistically. This "black" group looks fairly natural and facultative, like it has done well to establish a colony and exploit this ecological niche. In your photograph, the black sheath (mantle) looks quite confined. Usually people just suppose that their roots were stained by the soil, whereas in fact this is more-often than not, a distinct microbial community. It is both a different species and a separate colony than the "orange" group.

The "orange" group seems to have less preference for the older section of the root, and it is clearly more dependent upon the growing portion of the root, confined to a root horizon as opposed to colonising the root epidermis (or other locations the aquarium soil). This means that it is in an facultative association with the plant. This is probably due to the abundance of root nutrient exudates being produced by the plant and going into the aquarium soil surrounding it. Plants will typically exude nutrients including sugars, amino acids, organic acids, vitamins, and high molecular weight polymers in order to regulate and promote their microbiome. This seems to be a tactic being employed by your plant. It is both feeding the good guys whilst inhibiting the bad guys. Clearly it is a strategy that is working out because this root is massive, and it has not felt the need to branch out as a defensive response, which is a good sign.

We don't know whether either of these groups are mycorrhizal because we do not know whether they are in a mutualistic association with the plant. Some plants are nonmycorrhizal. Such plants can still get colonised, but they do not benefit in return. You would have to check <here> to see whether the family of plants your are growing fits into that category. Of the mycorrhizal associations, there is one that stands out as forming a similar black sheath (mantle) and it falls into the category of ectomycorrhizal fungus (abbreviated to ECM, see here). However, many of these symbiotic ECM associations tend to form on  newer "growing" roots as opposed older ones. We don't see that in your photograph, but in case you do, this is what a "black" ECM would look like:







JoshP12 said:


> I had a hunch it was the rhizosphere but had never “seen one”.


Technically I shall refer to your tank from now on as the UKAPS unofficial rhizotron


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## dw1305 (30 May 2022)

Hi all,


JoshP12 said:


> What are all these crazy colours!?


I' m pretty sure the orange zone, around the root, is a zone of iron (Fe) III oxide. These zones form because of a combination of root exudates and bacteria in the rhizosphere.

Iron is a bit of a Jekyll and Hyde plant nutrient, it is incredibly abundant in the soil, but unavailable in oxidising conditions, and plants really struggle to obtain the iron they require. 

Where you have reducing conditions the tables are turned and the plant needs to immobilise the the ferrous (iron II) ions to avoid toxicity issues.

Cheers Darrel


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## Simon Cole (30 May 2022)

dw1305 said:


> I' m pretty sure the orange zone, around the root, is a zone of iron (Fe) III oxide


An example would be from <here> <open source (uncorrected version)>:


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## Hanuman (30 May 2022)

JoshP12 said:


> For reference:


That is some serious substrate thickness! Planning to grow a pine tree?


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## JoshP12 (30 May 2022)

Hanuman said:


> That is some serious substrate thickness! Planning to grow a pine tree?


I don’t know what I’m growing!!! 😂


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## JoshP12 (30 May 2022)

Looks like I have the iron oxidation further “to the right” @dw1305 @Simon Cole






Maybe this is the orange living thing:





And around the other side the same horizon:


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## _Maq_ (12 Jul 2022)

What about sulfur? Sulfate reducing or sulfide oxidizing bacteria?


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## JoshP12 (13 Jul 2022)

Minor update - all of the orange turned black … .


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## _Maq_ (13 Jul 2022)

JoshP12 said:


> Minor update - all of the orange turned black … .


Iron sulfide?


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## JoshP12 (14 Jul 2022)

_Maq_ said:


> Iron sulfide?


Nice idea — any thoughts about the “nature” of these bacteria?

What I mean is: could there be a correlation with matureness (whatever that means) of the ecosystem? 

Or any indication of relative oxygen concentrations?


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## Simon Cole (15 Jul 2022)

When iron (Fe) III oxide reduces chemically (perhaps due to lower oxygen levels), you tend to see iron (Fe) II oxide which has that distinct black colour.
My guess is that these orange bands were formed due to roots carrying oxygen down from the emergent stems, which subsequently changed colour when the roots died and the source of oxygen ran out.


 < iron (Fe) II oxide.

We do not always see such beautiful iron (Fe) III oxides in or aquariums because few of us bother to allow plants to emerge from the water into the air. It is clear that this practice provides massive amounts of oxygen to the root zone.
I still suspect that you had some sort of microbial associations going on. I am now fairly convinced that you had  iron-oxidising bacteria present. The fact remains that there was an orange inhibited zone around some of your roots in your first photograph, and no orange staining on the roots. If it was mineral root horizons, then I would expect to see something rather different. So I still think that this is due to facultative bacteria in association with your plants. If it was iron-oxidising bacteria, then remember that although they are deriving energy from the iron oxides (in the same way that we respire with oxygen), that they will be obtaining the bulk of their organic molecules from the root exudates provided by your plants, or the soil itself.


 < gelatinous slime mass of iron-oxidising bacteria in a surface water.


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## dw1305 (15 Jul 2022)

Hi all,


JoshP12 said:


> Minor update - all of the orange turned black …





Simon Cole said:


> When iron (Fe) III oxide reduces chemically (perhaps due to lower oxygen levels), you tend to see iron (Fe) II oxide which has that distinct black colour.
> My guess is that these orange bands were formed due to roots carrying oxygen down from the emergent stems, which subsequently changed colour when the roots died and the source of oxygen ran out.


Same as @Simon Cole for me.


Simon Cole said:


> I still suspect that you had some sort of microbial associations going on. I am now fairly convinced that you had iron-oxidising bacteria present. The fact remains that there was an orange inhibited zone around some of your roots in your first photograph, and no orange staining on the roots. If it was mineral root horizons, then I would expect to see something rather different. So I still think that this is due to facultative bacteria in association with your plants. If it was iron-oxidising bacteria, then remember that although they are deriving energy from the iron oxides (in the same way that we respire with oxygen), that they will be obtaining the bulk of their organic molecules from the root exudates provided by your plants, or the soil itself.





Simon Cole said:


> It is clear that this practice provides massive amounts of oxygen to the root zone.


<"and again">.


dw1305 said:


> The uncertainty about what is happening in the sediment was one of the reasons for mainly talking about what might be happening in the <"rhizosphere"> the zone of fluctuating REDOX values, where exudates from the root will be altering the physical, chemical and microbial composition of the substrate.
> 
> In the <"Influence of submerged macrophytes, temperature, and nutrient loading on the development of redox potential around the sediment–water interface in lakes"> it mentions that their experimental plants didn't have much radial oxygen loss.
> 
> I'd guess that if they had used _Cyperus_ spp, <"_Phragmites australis_">, _Nelumbo_ sp. or an <"Amazon Sword"> (_Echinodorus_ spp) etc. that their findings may have been slightly different.


cheers Darrel


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