Life in the litterbox

When life gives you lemons, you make lemonade. And when life gives you kitty litter, you make… a new affordable growth system that could make it easier to broaden our understanding of plant-microbe interactions!

Most of us ‘big things’ (people, plants, monkeys, millipedes), tend to function so well in this world of ours because we have a whole bunch of ‘little things’ (bacteria and viruses) living on or around us.

In fact, it may be argued that big organisms should be seen less as individuals, and more as holobionts – a metaorganism of host and microbiota. One big thing. Millions of tiny things.

We’ve known for a very long time, for example, that many plants rely on rhizobacteria to fix their nitrogen, and on mycorrhizal fungi to mobilize soil phosphates.

For plants, microorganisms not only help aid nutrient uptake and promote growth, but also work to protect our green friends against a variety of biotic and abiotic stresses. As such, it’s perhaps unsurprising that scientists have proposed that synthetic microbiota might be developed to help support plant growth (and productivity) under different environmental conditions.

Of course, in order for this to work, we need to have a thorough understanding of both plant-microbe, as well as microbe-microbe interactions. Past research that ranged from 16S rDNA sequencing to meta-omics, studies, to the entire development of a culture collection covering the bacterial species associated with Arabidopis, have revealed that the plant microbiome is diverse and complex. But more than that, it’s also structured, meaning that the effects of adding different microbes to a mix can’t easily be predicted. For example, while both Pseudomonas fluorescens A506 and Pantoea vagans C9-1, can individually control the harmful bacteria causing plant fire blight disease, adding both at the same time actually limits the biocontrol effect. That is to say, a positive and another positive doesn’t always add up to a positive!

Ultimately, to understand the plant microbiota, we need to do what we always do in science – break the system to see how it works. We can either remove all of the microbes, and then see what happens as we add them, one by one. Or we can create drop-out systems, in which everything is included except for our one microbe-of-interest.

Of course, in order to do this, we need so-called ‘gnotobiotic’ systems. Growth conditions which are sterile, except for the specific microorganisms that the scientists have chosen to add in.

Typically, gnotobiotic growth of lab plants is undertaken in jellified plant nutrient medium (agar), or alternatively, in hydroponics (the roots dangle in water) or aeroponics (the roots sit in the air and are sprayed with water). These systems have two main problems, the first being that root development doesn’t really resemble that seen in soil (and therefore in crops in fields), and the second being that all systems are highly humid. As it turns out, humidity is one of the defining factors that drives bacterial colonisation. By contrast, soil itself, is absolutely teeming with bugs (the microscopic kind), and although these can be sterilised away by baking, this also alters the structure and geochemical properties of the soil.

(By the way, we’ve previously written about how scientists have tried to better mimic field conditions by developing soil-like growth substances that also happen to be see-through.)

So, in order to develop a new system to observe plant-microbe interactions, Moritz Miebach and colleagues at the University of Cantebury, New Zealand turned to zeolite. Zeolite is easy to sterilize, porous, allows root aeration and has excellent water and nutrient holding capacity. It’s previously been tested in other plant systems, and even used on some plant-space missions.

Oh, and it’s easily and affordably sourced. From kitty litter.

The authors called their system ‘litterbox’.

To test how well plants grew in the litterbox, the scientists did side-by-side comparisons of agar- and zeolite- grown arabidopsis. Generally, plants were green and healthy on both media, and showed a similar rate of growth. However, litterbox plants grew more reproducibly – there was less variation from plant to plant. Furthermore, they had roots that grew more, and curled less than those in the agar.

In short, they looked and acted more like the roots of soil-grown plants.

Fluffy had regrets about letting those scientists adopt him.

The scientists also did a few more tests. They placed a silicon sheet (polydimethylsiloxane to be precise) on top of the zeolite, which created some physical separation between the microbiota of the plant aerial tissue, and that of the roots. They tested the bacterial carrying capacity of the aerial tissue microbiome (phyllosphere), and found that the amount of bacterial found on the zeolite plants was also closer to that of soil-grown plants than agar plants were. Additionaly, they did a couple of experiments that showed that they could alter the microbiome of the phyllosphere and the rhizosphere (below-ground) semi-independently.

All up, the scientists developed a system that is easily available, affordable and easy to establish. With just a bit of Purrfit Clay kitty litter and a few other ingredients, low variation consistent plant growth could be acheived, producing plants that better resemble ‘real life’ than those grown on agar. Furthermore, the Litterbox system allows for easy control of humidity, light and temperature. All in all, a little bit of kitty litter might just turn out to be a cool tool to advance microbiota research!

References

Miebach M, Schlechter RO, Clemens J, Jameson PE, Remus-Emsermann MNP. Litterbox-A gnotobiotic Zeolite-Clay System to Investigate Arabidopsis-Microbe Interactions. Microorganisms. 2020;8(4):E464. Published 2020 Mar 25. doi:10.3390/microorganisms8040464

1 thought on “Life in the litterbox”

  1. Pingback: Let’s get Fascinated – Plants and Pipettes

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