Tag Archives: Microbes

Pathogens – nasty tree microbes

A pathogen is a microorganism such as a virus, bacterium, oomycete (water mould) or fungus which causes disease and/or death.^ref Examples you might have heard of include Dutch Elm disease (caused by the fungus Ophiostoma ulmi), Horse Chestnut bleeding canker disease (caused by the bacterium Pseudomonas syringae v. aesculi), Ash dieback (caused by the fungus Hymenoscyphus fraxineus), Sudden Oak Death (caused by the oomycete Phytophthora ramorum) and mosaic viruses in vegetables.

The groups of pathogens causing forest tree diseases and their prevalence is shown in the chart below. 85% of these are fungi and the remainder are divided among bacteria, viruses, nemotodes (microscopic worms) and oomycetes. Oomycetes appear similar to fungi but are actually not genetically similar, and are classed in the Chromista kingdom.^ref

Link to source article here; original sources Butin, 1995; Capretti and Ragazzi, 2010; Manion, 1991; Tainter and Baker, 1996

Note that as they are not microorganisms, insects are not classified as pathogens, although they can also cause significant damage and death to trees. Insect damage is a subject for another post, but for this one, their main role is in introducing pathogens to trees by delivering microbes on them into wounds they create (this is called being a pathogen ‘vector’).

Every living thing is in a battle for survival so that its genes can be passed on to future generations. This means that both trees and pathogens are constantly evolving to outsmart each other. But fungi and bacteria can also be beneficial and even necessary for trees, so you can’t just try to kill off every fungus or bacteria as this will destroy the ones that trees actually want and need.

The other thing to keep in mind is that plants have a completely different ‘lifestyle’ to animals in the sense that they are plastic and can grow new tissues, synthesise new compounds and respond to pathogens in a very different way to humans. As noted elsewhere, in response to wounds, trees ‘don’t heal they seal’ and they don’t have an immune system in the same way as animals.

But, there are three ways to help your trees avoid damage from pathogens. The first is to prevent them from being exposed to pathogens in the first place. The second is to remove any pathogens which do take hold. The third is to bolster your trees’ defences so they can fight off the pathogen and its effects.

How can you prevent exposure to pathogens? A tree’s first line of defence is its bark, leaf cuticle, and the pectin and lignin in cell walls, which are physical barriers which prevent pathogens from entering its cells.^ref Since they can’t run away, this is the main way that trees avoid exposure. But in bonsai we do a heck of a lot of pruning, which unfortunately breaks the physical barrier, leaving the tree vulnerable.

To minimise the chance of nasty microbes attacking your tree as a result of pruning there are some steps you can take. Firstly, practice excellent hygiene and make sure your pruning tools are disinfected regularly, particularly when moving between trees. But be careful that the disinfecting method you use doesn’t damage your tools (carbon steel is particularly vulnerable). Soap and water can work (but dry off the water), or you could use an antimicrobial oil such as tea tree oil (from Melaleuca alternifolia) or oregano oil (from Origanum vulgare) – not only are these antimicrobial^ref1,^ref2 but they also protect steel from corrosion.^ref1,ref2

Another approach is to prune during the wintertime. The latter helps because in general most living things are more active in warmer weather – including pathogens. Pruning in winter reduces the likelihood that a pathogen will enter a wound before the tree can seal it off.^ref Also many fungi and oomycetes prefer a moist environment, so when pruning try to avoid leaving the wound wet. Angling pruning wounds towards the sun can also be beneficial, since sunlight has disinfecting as well as drying properties (although probably not so much during the wintertime in higher latitudes).^ref

Contrary to some advice, wound sealants have not been shown to reduce bacterial or fungal infection on tree wounds.^ref This is because a tree wound is not sterile so any sealant can seal pathogens in as well as out. One wonders whether applying an antimicrobial oil to a wound might work, but I cannot find any studies looking into this. Recently I tried applying raw linseed oil to the cut ends of various crabapple branches I was trying to propagate, but these ended up with large communities of mould on them regardless (possibly because I was using high humidity which is perfect for fungal growth).

One interesting pathogen avoidance method is to decouple the seasonal timing between the host plant and the pathogen vector (vector means the delivery method of the pathogen, often an insect). One study found that Dutch Elm disease was avoided by trees which flushed early, since they were not as susceptible to infection after this point.

The second way to help your trees avoid pathogens is to remove them once there. This is tricky since by definition a pathogen is microscopic and impossible to see with the naked eye. You can’t go and squash every bacterium on your tree! It is possible to kill pathogens using antibiotic and/or antifungal substances, but effectiveness varies depending on what you need to remove and usually involves unpleasant and toxic chemicals (such as glyphosate) which also kill good microbes.

Biological control is an alternative to chemicals, this means finding another organism which feeds on or somehow damages the pathogen in question. An example of biological control is the introduction of the Myxoma virus into Australia to control the rabbit population. In trees, the fungus Trichoderma is used as a biological control agent, and it is found in bacterial inoculants for plants such as this one. Species of Trichoderma have been found to be effective against Armillaria root rot (also known as the dreaded honey fungus) and pine pitch canker.^ref

The final way to help your trees is to bolster the natural processes they use to resist the negative effects of pathogens. Plants produce a huge range of substances which have defensive effects, and can detect pathogens with surprising speed and specificity. When a pathogen is detected by a plant, it first activates a specific ‘pattern-triggered immunity’ response which is believed to be sufficient to defend against a wide range of pathogens.^ref This is known as ‘basal resistance’. A second line of defence detects the so-called ‘effectors’ – substances that pathogens create to avoid the pattern-triggered response. This second ‘effector-primed immune response’ causes cell death at the site, limiting the spread of the pathogen^ref and is known as the ‘hypersensitive response’. Plants also synthesise a wide range of defensive compounds such as resins in conifers, terpenoids or essential oils, saponins and flavonoids (of which 9,000 are known).^ref1,ref2 These help them deter pathogens by making their cells poisonous or unpalatable.

So ensuring your plant is not stressed by lack of water, light or nutrients is one way to help it have the resources to defend itself. Another way is to provide it with beneficial microbes such as mycorrhizal fungi and beneficial bacteria. One study found that providing the bacteria Bacillus cereus to tomato plants enhanced their resistance to pathogens by activating the plant growth hormones salicylic and jasmonic acids. Cultivating a healthy rhizosphere (root microbiome) which supports your tree’s health can be achieved by using a well-aerated soil mix and by not constantly repotting. Repotting risks losing the microbiome which a tree has built up over time, for this reason I always try to add back in some of the previous soil when I repot.

So what should you be doing as a bonsai enthusiast to avoid any nasty pathogens ruining your great work? I’d suggest three things. The first is good pruning practices – minimising wounds, avoiding pruning wounds becoming wet or humid, and vigilance in disinfecting tools and pots. The second is to keep your trees vigorous and healthy – particularly before doing large-scale pruning or defoliation. Give your trees lots of water, nutrients and sunlight to help them bolster their defences. And finally help your trees out with the addition of beneficial fungi & bacteria; products containing these can be found online.

The Endosphere

Although it might sound like we’re veering into science fiction territory, the endosphere is actually part of a plant’s microbiome, like the rhizosphere and the phyllosphere. It is the community of microbes which live inside the plant itself – that is, between and in its cells. It’s only in the last few decades that research on the endosphere has accelerated – this has found that in fact a wide variety of microbes including bacteria and fungi live inside plants for at least a part of their lifecycle.^ref They are known as endophytes – and some of these are symbiotic whilst others can be pathogens.

Endophytes are found throughout the plant, in leaves, roots and stems, in spaces between cells as well as within cells themselves; the greatest number are found in roots, then leaves, then fruit/flowers. The types of microbes in residence depends on the microenvironment in each part of the plant, the specific physical and chemical characteristics in each environment attract different microbes.^ref

To enter the plant in the first place, microbes come from outside, through the root tips and hairs, through stomata and trichome pores in leaves, fruit & flowers, through holes in the stem made by insects, or by producing enzymes which break down plant cell walls to create an opening. Often these microbes are present in the rhizosphere or phyllosphere, and they migrate into the plant for all or part of their lifecycle.^ref Usually they live between cells, but some examples of bacteria and fungi entering plants cells have also been found. Endophytes can be transmitted vertically (from mother plant to seed), and horizontally (from the outside environment).^ref

Of all the spheres, the endosphere is the hardest to study, so there isn’t a huge amount of research which demonstrates what endophytes actually do when they are inside plants and how the host plant might benefit. Some findings are that endophytes are able to detect Reactive Oxygen Species (“ROS”) and may be able to help plants fight high ROS levels (eg. acting as an anti-oxidant).^ref Others have found endophytic fungi which produce the plant growth regulators gibberellic acid and indole acetic acid (auxin), and that this contributes to greater root & shoot mass.^{ref1 ref2} One study found an endophyte which conferred resistance to Dutch Elm Disease in vitro^ref. Finally a large number of endophytes associated with trees have been found to produce Taxol^ref, the best-selling cancer drug ever manufactured^ref and this promises to be a way for greater volumes of the drug to be created.^ref So like bacteria & fungi across the microbiome, these microbes appear to be pop-up pharmacies within the tree.

The endosphere probably doesn’t need to be your prime concern from a bonsai perspective. Like the other components of the tree’s microbiome, you want to foster a healthy one, which benefits the tree, and not an unhealthy one. Doing this mainly involves not killing them off!

The Phyllosphere

The phyllosphere is the community of microbes which live in and on a plant’s leaves. I had no idea that this even existed before writing the section for this website about the microbiome. Of course, if you think about it for a microsecond, it must! Our world has more microbes than anything else by several orders of magnitude, so, there must be microbes in a tree’s leaves. But the phyllosphere has been less publicised due to the intense interest in the rhizosphere (root microbiome) and in its beneficial microbes which can help plants grow by manipulating the soil and root environment.

The phyllosphere is different to the rhizosphere in that its main microbial members are bacteria and not fungi, although fungi are present, along with some archaea. It has been estimated that there are 1 million -10million bacterial cells per cm² of leaf surface.^ref And worldwide, the phyllosphere is an important microbiome, with a possible 10²⁶ cells! But it’s a relatively hostile environment, with fluctuating temperature & humidity and limited nutrients on the leaf surface. The shape and structure of the leaf at a microscopic level provides a range of microhabitats for bacteria, including the bases of trichomes, stomata, hydathodes (leaf pores), grooves along the veins, epidermal cell junctions, and cuticle depressions.^ref A study into tree phyllospheres found 129 bacterial species were significantly associated with the gymnosperms including Armatimonadetes, Actinobacteria, Bacteroidetes, Acidobacteria, TM7, TM6, Deltaproteobacteria, OD1, Fusobacteria, and FBP and 79 with the angiosperms including Chlamydiae, Proteobacteria, Gammaproteobacteria, Alphaproteobacteria, and Firmicutes.^ref

Bacteria on a leaf surface, from: https://www.ethlife.ethz.ch/archive_articles/090915_blattleben_kw/index_EN.html

What determines the microbial mass and mix on leaves is a combination of different factors, including the nitrogen content of leaves, the specific leaf area (related to carbon availability), wood density and seed mass^ref and the largest part of the variation seen between phyllospheres comes down to the host species. Conifers have a different phyllobiome than other species, for example they have less ice nuclei active bacteria (bacteria which can cause ice crystals to form) and they have Frankiaceae which is involved in nitrogen fixing in the soil.^ref Location also plays a role, with urban trees displaying a different phyllosphere makeup – correlated to ultrafine particulate matter and black carbon on the leaves.^ref

Bacteria usually require an available carbon source. You might be surprised to know that similar to roots, leaves also produce exudates (substances they exude into the environment). These include a wide range of carbon compounds, such as carbohydrates, amino acids, organic acids, and sugar alcohols, primarily products of photosynthesis, as well as proteins, oils, secondary metabolites and mucilage.^ref These carbon sources are not the only ones – the Methylobacterium species can use methanol exuded from the leaf from the breakdown of pectin as its only carbon source.^ref One of the bacterial families found on birch – Rhodospirillaceae – is able to photosynthesise, removing the dependence on leaf carbon sources. Another study discovered that certain phyllosphere bacteria can use diesel for their carbon source!^ref

Similarly, bacteria in the rhizosphere produce a range of substances just like they do in the rhizosphere – biosurfactants which reduce surface tension, degrade hydrocarbons and improve moisture levels and dissolved nutrients on the leaf surface, plant growth regulators which open up the leaf cells and cause them to leak nutrients, enzymes which help break down nutrients and protect the bacteria from solar radiation, and phytotoxins (if the bacteria is a pathogen).^ref

The benefits of phyllosphere microbes to their host are similar to those in the rhizosphere – for example Acetic Acid Bacteria have been found to perform nitrogen fixation within the needles of Pinus flexilis^ref, others confer resistance to Bursaphelenchus xylophilus-induced pine wilt disease^ref, some phyllosphere fungi produce zeatin, a cytokinin (plant growth regulator)^ref and others auxins, some also produce anti-freeze proteins which lower the freezing temperatureon the leaf.^ref Bacteria are implicated in the bioremediation of harmful chemicals or pollutants^ref, improved tolerance to stress, production of proteins which trigger the plant to mount defences against pathogens as well as those which attract populations of beneficial fungi.^ref

So, just like the rhizosphere, the phyllosphere is a very active place with many microorganisms playing different roles and constantly interacting in a dynamic ecosystem. What this means for bonsai is that there likely are organisms in the foliage which benefit your plant. Similar to the advice in general around the microbiome, applying fungicides, anti-bacterials and chemical pesticides can kill phyllosphere organisms so avoiding this is a good idea.

Biochar

(Thanks to Dr. Karen O’Hanlon of Probio Carbon for answering some of my questions about biochar).

Biochar is a product which has been advertised as a beneficial component of bonsai soil over recent years. So what exactly is it?

Biochar is basically charcoal which has been “produced from organic waste using pyrolysis technology under temperatures ranging from 400C to 700C where oxygen is either absent or depleted”.^ref Pyrolosis means decomposing carbon-based materials through the application of heat.^ref So a feedstock (source material) is acquired and heated in the absence of oxygen for a given period of time to create what you would probably recognise as charcoal. The structure of biochar is shown in the image – as you can see, it has many, many holes in it.

Scanning Electron Microscope image of biochar
https://www.rhs.org.uk/soil-composts-mulches/biochar

So why would you add biochar to your bonsai soil? There are a few good reasons. It has been proven to improve water availability^ref, act as a fertiliser reducing the need for chemical fertilisers^ref and increase microbial biomass^ref (ie. it attracts beneficial microbes).

An experiment conducted in Colchester, UK by the Bartlett Tree Research & Diagnostic Laboratory amazingly found that ash trees treated with biochar did *not* get infected by ash dieback disease over a period of 4 years even when the disease was present in adjacent trees on the same site. They believed the reason for this was that the biochar enhanced the trees’ immune system and improved root growth.^ref

The microbe aspect of biochar is really interesting – in one study it was found that microbes living in it were able to ‘mine’ the biochar pores for phosphorus. So it appears to have synergy between its composition (with nutrients for plants) and its attractiveness to microbes which can help get those nutrients into plants.

One of the key physical properties of biochar is that it has a massive surface area, relative to its size – in one study on malt spent rootlets (a residue from brewing) it was 340 m² per gram!!^ref That’s larger than the size of a tennis court for every gram of biochar.^ref This increased surface area along with the physical structure of biochar having lots of tiny pores, results in greater water retention in the soil.^ref

Biochar can be made from basically any organic material, from forestry to food production to agricultural by-products and this source material is the main determinant of its chemical properties.^ref So when choosing a biochar for your bonsai soil, you want to know what it has been made from, and what this means in terms of its properties. Some of the properties which vary significantly include pH, surface area and cation exchange capability/electrical conductivity. For bonsai I would say you want high surface area & pore volume (to assist with water availability) and high microbial mass. The fertiliser aspects are probably a nice-to-have. Looking at the table below this means probably biochar made from a wood-based source material is best.

There is quite a bit of research out there on different biochar properties, which I will summarise here for you to read through. Unfortunately I haven’t found any research which looks at volume of microbes for each feedstock, but I would expect this to be positively associated with surface area.

Biochar Feedstock	Properties
Wood	Highest surface area (leading to better water retention) and highest pore volume (a factor of 10 higher than manure) Lowest cation-exchange capability Largest amount of C Contain less plant-available nutrients More electrical conductivity Lowest ash content (associated with lower pH) Micro-nutrient content mixed (see table here) Total bioavailable nutrients mixed (see table here)
Crops & grasses	Highest average particle size Highest K content Lowest calcium carbonate equivalents Micro-nutrient content mixed (see table here) Total bioavailable nutrients mixed (see table here)
Manure	Lowest surface area and lowest pore volume Highest cation-exchange capability Highest calcium carbonate equivalents Lowest average particle size Highest ash content (associated with higher pH) Greatest N, S, P, Ca, and Mg concentrations Highest micro-nutrient content (Fe, Cu, Zn, B, Mn, Mo, Co, Cl) Total bioavailable nutrients mixed (see table here)

Source: https://link.springer.com/article/10.1007/s42773-020-00067-x/tables/1

The temperature at which the biochar is created makes a difference too. Increasing pyrolisis temperature leads to “increased biochar C, P, K, Ca, ash content, pH, specific surface area (SSA), and decreased N, H, and O content”^ref

Like many things in life though, you can have too much of a good thing. In some studies, too much or the wrong biochar in soil has led to phytotoxicity^ref You might also be wondering why it doesn’t just remove all the nutrients in the soil like activated carbon, which is used in aquariums and drink bottles to remove metals, chlorine and contaminants. When asked this question Dr. Karen O’Hanlon at Probio Carbon said it was because biochar is not ‘activated’ to the same degree as activated carbon. Reading more about this, the absorbent properties of biochar are “1/6th to 1/12th that of high quality activated carbons”.^ref Activation forces more pores and surface area into the charcoal, this is done by varying the temperature and pyrolysis process. So whilst there probably is some nutrient absorption, it’s not going to be at the same level as activated carbon and can be compensated for by the nutrients within the biochar themselves and the increased microbial activity.

Bonsai Tree Growth Stages

Most bonsai trees progress through stages of development, each with a different objective. In general the progression is thicken trunk -> achieve branch & root structure -> achieve branch, foliage & root ramification -> reduce leaf size -> evolve as branches grow/fall off. The faster we can move through the first few development stages, the faster we will have beautiful, well-proportioned bonsai – harnessing the tree’s natural growth is a way to speed this up. We also want to avoid doing things which slow down a tree’s growth during these phases, as this will mean it takes longer to get the tree we want. Read about how trees grow before starting at #1 below. Also consider what do old trees look like?

1. Trunk

Some bonsai enthusiasts collect mature trees for bonsai specifically so they can start with a thick trunk, following a collection process which minimises damage to the tree. The alternative is growing your tree’s trunk. Once a tree has its roots and foliage reduced in size in a bonsai pot, it won’t generate the energy needed to make significant sapwood additions and its girth will only increase by small increments every year. So you really need to be happy with the trunk size first before you stick it in a tiny pot. But – how big should a bonsai tree’s trunk be?

2A. Branch Structure & Overall Shape

Arranging the branches is what gives you the canopy and overall foliage shape that you’re after and the first step in this process is growing (or developing) the branches you want in the positions they are needed. Growing a branch starts with a new bud, which, unless it’s a flower bud, becomes an extending shoot and eventually a new branch. So firstly you need to work out where new buds will grow on your tree and then deal with the extending shoots as needed to get the required internode length.

You may need to remove some buds and shoots if they don’t help achieve the shape you are looking for – this should be done as soon as possible to avoid wasting the tree’s finite energy reserves. You have a trade-off to make here because leaving more foliage on the tree will provide more energy overall which contributes to its health and ability to recover from interference. However, growing areas of the tree which won’t be part of the future design is a waste of energy. You don’t want to remove so much of the tree’s foliage that it struggles to stay alive or develop the areas that you do want to grow out.

When you are creating your branch structure, often you will need to reposition branches – this is done with a wide range of different tools and techniques. A more advanced technique for adding new branch structure is grafting.

Sometimes the trunk itself or larger branches need a rework, to make them more interesting or to make them look more like old trees – for example adding deadwood or hollowing out the trunk. Usually this is achieved through carving.

2B. Creating a Strong Root System

The trunk thickening and branch structure phases both work best when the tree has lots of energy and so letting it grow in the ground or in a decent sized pot during these phases will get you there quickest. This also allows the roots to keep growing, but you want to understand about the role of roots, and root structure & architecture even if you still have your bonsai in a training pot. Particularly in this case, knowing about how to foster the the rhizosphere will help your tree stay vigorous. To maximise the roots’ exposure to nutrients and water you want to encourage Ramification of Roots (lateral root development).

Eventually it’s time to move the tree into a bonsai pot. This requires cutting back the roots, but as long as the roots are balanced with the foliage in terms of biomass, the tree should be OK. Root growth is usually prioritised outside of times of stem/foliage growth, and above 6-9 degrees C. So repotting might be best conducted at times that meet this criteria. Your growing substrate/medium is an important consideration.

3. Ramifying Branches & Foliage

Ramification is when branches subdivide and branch, giving the impression of age and a full canopy – and a well-ramified tree is a bonsai enthusiast’s goal. There are some techniques for increasing the ramification of branches and foliage. But not as many as there are for root ramification.

This stage also involves ongoing branch selection and reshaping (see 2A above). Another consideration is whether to keep or remove flower buds.

4. Reducing Leaf Size

An end stage in the journey to bonsai perfection is leaf size reduction. In nature, leaf sizes reduce relative to the biomass of the tree as it ages but since bonsai are small this effect doesn’t translate since the biomass never gets large enough. The tried and tested method for reducing deciduous tree leaf size is actually to practice one of the various methods of defoliation. A couple of others are covered here in reducing leaf size.

When to conduct these various activities depends on when the tree can best recover from them – which is a function of the Tree Phenology (or Seasonal Cycles).

5. Evolving Branches

Trees are not static organisms – they obviously continue to grow which is what we harness in the above steps. Part of this is that eventually branches may become too large for the design, or they may fall off (Peter Warren notes that Mulberry are known for this). As bonsai artists we want to have this in mind so that branches are being developed which can take their place in the future. This is an ongoing version of step 2A.

Root Exudates

I had never heard of root exudates before creating this website, but in fact their production is so important to plants that they “invest up to 20–40% of their photosynthetically fixed C”^ref in this process.

Root exudates are basically substances created by root cells and sent out into the nearby environment – known as the rhizosphere. These can be waste products which diffuse across the cell wall, or manufactured compounds which serve a specific purpose in response to the environment.^ref

There are many different exudates produced by plants, including carbohydrates (sugars), organic acids (such as acetic, citric or malic acid), amino acids, flavonols (molecules which can have protective effects on cells), enzymes (such as amylase, which helps to digest carbohydrates), plant growth regulators (substances which stimulate cell growth, such as auxins), phenolic acids (which have anti-oxidant properties), flavonoids, terpenoids, tannins, steroids and an assortment of other substances^ref1,^ref2.

The roles they play are just as diverse, including:

producing food in the form of carbon metabolites to support beneficial bacteria (such as nitrogen fixing bacteria), fungi, nemotodes and protozoa^ref1,^ref2, which in turn assist with nutrient uptake and the production of their own compounds such as phytohormones
producing phytotoxins (plant poisons) such as terpenes (also found in conifer sap) to repel pathogenic microbes, invertebrate herbivores and parasitic plants^ref1,^ref2
changing the pH of the surrounding soil^ref
detecting ‘kin’ (related plants) and avoiding competing plants which are not related^ref
changing the soil chemistry to allow for better nutrient uptake – for example exuding chelating substances which allow for better uptake of metallic micronutrients, or organic acids which enable better phosphorus uptake^ref

So it seems that the roots of a tree can act like a sort of pharmacy, creating compounds that protect and nurture the tree and its beneficial partners via the rhizosphere. In my opinion what this means for bonsai is that firstly you don’t want to damage the tree’s ability to produce exudates, and secondly you don’t want to remove too much of the soil from a healthy root ball.

You can damage the tree’s ability to produce exudates by underwatering/drought – and this may not be recoverable^ref, but also by failing to provide all of the nutrients and micronutrients needed for healthy growth (by not fertilising enough).

What’s in the bonsai pot is clearly more than just roots and soil – it’s an entire ecosystem delicately managed by the tree itself. So perhaps being less heavy-handed during repotting would be a good idea – replacing a good amount of the soil back into the pot along with its microbes, exudates and adjusted chemical makeup.

The Rhizosphere

Roots exist in a their own ecosystem along with soil, chemical compounds, microorganisms and variations in pH, humidity and temperature. This environment is known as the ‘rhizosphere’, a term created by Lorenz Hiltner in 1904, using the greek word for root ‘rhiza’.

The term refers to the area around the roots, and is broken into three parts. “The endorhizosphere includes portions of the cortex and endodermis in which microbes and cations can occupy the “free space” between cells (apoplastic space). The rhizoplane is the medial zone directly adjacent to the root including the root epidermis and mucilage. The outermost zone is the ectorhizosphere which extends from the rhizoplane out into the bulk soil.”^ref

The rhizosphere is FULL of microbes – this article^ref estimates there are 1000-2000 times the number which are found in non-rhizosphere soil. These include endomycorrhiza and ectomycorrhiza as well as beneficial (and pathogenic) bacteria. Below is an estimate of the number of genes represented in a sample rhizosphere across each type of organism (a list of the species included are in the research paper^ref)

https://academic.oup.com/view-large/figure/90643206/fmr12028-fig-0001-m.jpeg

Rather than passively respond to the rhizosphere, roots produce ‘exudates‘ – substances released from their cells – which are used both to sense the environment (such as, where competing roots are located and the presence of beneficial microbes and nutrients) and to alter it to the plant’s benefit. So the rhizosphere is a very dynamic place, teeming with life and being constantly manipulated by the tree for its own benefit. Below is a great image illustrating everything that’s going on – different mycorrhiza, bacteria and the roots interacting in the rhizosphere.

Plants, Mycorrhizal Fungi, and Bacteria: A Network of Interactions
Paola Bonfante and Iulia-Andra Anca
Annual Review of Microbiology 2009 63:1, 363-383

‘Mycorrhiza’ are fungi which have a symbiotic relationship with roots – they each provide something of value to the other party. The word comes from the Greek words for ‘fungus’ and ‘roots’ so one should strictly call them mycorrhiza and not mycorrhizal fungi since the latter is an example of ‘RAS syndrome’ (redundant acronym syndrome, which itself is also an example of RAS syndrome).

According to one study, “for efficient nutrient uptake, most land plants need to be associated with mycorrhizal fungi that supply minerals, increasing their productivity and conferring resistance to stress.”^ref So these fungi are actually a critical part of life on earth, and necessary for healthy plant function.

Mycorrhiza are usually divided into two groups – endomycorrhiza and ectomycorrhiza.

‘Endo’ comes from the Greek ‘endon’ meaning ‘within’ – and endomycorrhiza (known as Arbuscular Mycorrhiza or ‘AM’) have hyphae (fungal threads) which actually penetrate the plant’s root cells and establish an intracellular symbiosis with the plant^ref. AMs scavenge for nutrients such as Phosphorus and Nitrogen released by saprotrophic microbes (ie. bacteria which feed off dead material) and make these available to the plant.^ref

‘Ecto’ comes from the Greek ‘ektos’ meaning ‘outside’ – and ectomycorrhiza (‘ECM’) form a thick mantle around root tips from which clusters of hyphae extend beyond the root zone.^ref They ‘mine’ Nitrogen and Phosphorus from the soil by producing enzymes which digest soil organic matter – they can then make these available to the trees in return for carbon sources such as sugars.

Whether a particular species of tree is associated with endo- or ectomycorrhiza is detailed in this site. The trees we’re interested in from a bonsai perspective fall in each camp: Associated with ECM are oak, beech, hornbeam, birch, hazel, alder (actually with both), tilia (lime/linden), chestnut and all of the Pinaceae family (including fir, cedar, larch, spruce, pine & hemlock). Associated with endomycorrhiza (AM) are grapevine, Prunus (cherry, peach, plum etc), pyrancantha, magnolia, Ilex (holly), Araucariaceae, wisteria, ficus, mulberry, ash, olive, all maples, horse chestnut, poplar/aspen, willow, buddleja, yew, camellia, elm, podocarps, flowering quince, hawthorn, apple, cotoneaster and all of the Cupressaceae family (including Cryptomeria japonica, cypress, junipers, redwoods and thujas),

Aside from this, azaleas are associated with a different mycorrhiza called ericoid.

Fungi aren’t the only microbes in the rhizosphere – it’s also teeming with bacteria – ‘rhizobacteria’. Symbiotic bacteria in the rhizosphere – known as Plant Growth Promoting Rhizobacteria (‘PGPRs’) deliver a raft of benefits to their host plants – some of which they literally could not survive without. They improve a plant’s resistance to pathogenic fungi, bacteria, viruses and nematodes as well as abiotic (environmental) stress like drought or heavy metal pollution, they also fix nitrogen into root nodules, convert organic nitrogen into inorganic forms (NH₄⁺ and NO₃^–) which are available for plants, improve the availability of phosphorus and iron, control other nutrients including sulphur, iron and manganese, and synthesise plant growth regulators which improve plant growth.^ref1, ^ref2 This study has a table showing some of the positive plant responses to specific bacteria in research studies.

They achieve these outcomes for their host plant partly by going about their task of decomposing organic matter, but crucially also by producing substances including siderophores which make iron available, enzymes which degrade the cell walls of pathogens, volatile compunds such as hydrogen cyanide, biosurfactants which lower the surface tension of liquids, antibiotics which target pathogenic bacteria and phytohormones which promote plant growth processes; all of these go into the soil and into roots.^ref Bacteria are also able to remove toxic metals from the soil through several different mechanisms and pathways.^ref

This is such a fascinating area – bacteria turn out to be tiny bespoke pharmacies available to plants to help them thrive. And plants are not just passive recipients of bacteria – they create root exudates which attract bacteria they specifically need at a point in time, they are able to manipulate the rhizosphere to meet their needs.^ref Plant genotype (ie. it’s genetic makeup) and the soil type are two main drivers that shape the rhizosphere microbiome.^ref pH is particularly important, with studies showing that bacterial diversity was highest in neutral soils and lower in acidic soils.^ref

The different bacterial species which are associated with different benefits for plants include the following^ref:

Plant Growth Promotion (supporting plant health & growth): Pseudomonas, Bacillus, Rhizobia, Achromobacter, Azotobacter, Arthrobacter
Biocontrol (fighting pathogens): Pseudomonas, Bacillus, Serratia, Pantoea, Acenetobacter, Xanthomonas, Alcaligens
Bioremediation (removing pollutants): Pseudomonas, Bacillus, Alcaligens, Arthobacter, Achromobacter, Azospirillum, Pantoea

On a final note, bacteria can produce ‘bad’ substances as well, particularly in anaeroic (no oxygen) conditions, when they produce phytotoxic nitrates and hydrogen sulphide. So avoid your bonsai substrate becoming too enclosed without aeration.

Roots

The roots of your tree are *just* as important as the above-ground parts, with a lot of responsibilities which aren’t immediately obvious. I’ve summarised the main ones here but there is a lot more detail in separate posts with links provided below. So why are roots so important?

they absorb water from the soil to meet all the tree’s needs (both for photosynthesis and transpiration)
they absorb all the nutrients that the tree needs from the soil (using a different process to water, hence a separate point)
they transport nutrients & water up to the above-ground parts of the plant, and photosynthates (the products of photosynthesis) down to the root tips
they produce exudates (secretions) which sense and control the rhizosphere (the environment in which the roots are growing)
they produce plant growth regulators for signalling and enabling growth within the plant
they store food for later use
they provide structural strength and stability for the tree by attaching it into the soil

Points 1 and 2 are fundamental to the health and growth of the tree – the roots are the mechanism for the tree to obtain all of the water and nutrients it requires (despite the mythic popularity of foliar feeding, this is only a way of augmenting nutrient absorption and not a primary mechanism). The mechanics of how they do this is described in more detail in how roots absorb water and nutrients – in summary it’s the fine roots and their root hairs which do the majority of the absorption since they have the closest and most expansive contact with the soil. There needs to be enough root surface area to supply the stems, shoots and leaves with the water and nutrients they require.

Point 3 reflects the fact that roots are part of a tree’s vascular system, that is to say, they transport the fluids necessary for growth around the tree. Above the ground the vascular system is present in stems, shoots and leaves, and below the ground it is present in the roots. Water and nutrients are transported up from the roots through the xylem and photosynthates (the products of photosynthesis) are transported down from the leaves and other storage organs in the tree via the phloem, to provide the energy and nutrients for the roots to grow and function. Do roots grow all year round? Find out here: when do roots grow?.

Points 4 & 5 show that roots are very much an active participant in tree growth and not simply a set of supply pipes. They produce both cytokinin and auxin (read more in the post about plant growth regulators), they also produce a huge variety of substances known as exudates which both sense and control the rhizosphere (the environment in which the roots exist). Researchers believe that roots use exudates to “regulate the soil microbial community in their immediate vicinity, withstand herbivory, encourage beneficial symbioses, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species”^ref. Read more about exudates and how they are produced in root exudates.

Point 6 reflects the fact that roots are used to store food, in the same way that the trunk and branches do this aboveground (throughout the ‘woody skeleton’ (Ennos)). I was going to tell you that a lignotuber is an example of this and show you a lovely picture of my eucalyptus, but then I read “contrary to common assumptions…the lignotuber in young eucalypt trees did not appear to be a specialized starch storage organ. Rather, the lignotuber resembled an extension of the stem because its starch concentrations and temporal fluctuations mirrored that of the stem.”^ref How roots store food and how much of a contribution to the plant’s overall storage capacity they make is debated. More on that in Root Food Storage (or, can I root prune before bud break?)

Finally as per point 7, the roots are responsible for physically holding the tree steady and stable against wind and gravity. They do this in many ingenious ways by adopting different root architectures – combining vertical taproots, lateral roots & sinker roots, creating ‘buttress’ roots, sending roots far from the trunk when needed and managing new root development in ways which stabilise the tree. More about this in root structure and architecture.

What all of this means from a bonsai perspective is that you need to pay just as much attention to the health and care of the roots of your tree as you do to the above-ground parts. Never mind developing a strong nebari for aesthetic purposes, you need to ensure that even though the roots of your bonsai trees are squashed into teeny-tiny pots, they are still able to perform the vital functions outlined above. Neglecting the roots will negatively affect the overall health of your tree.

Practically speaking, this is why you should aim to develop a well-ramified fine root ball, to provide the tree with lots of root surface area for nutrient & water uptake – taking into consideration the amount of biomass above-ground as this will determine how much root mass is needed.

The growing medium plays a huge role as well – this is your tree’s rhizosphere. It should provide the water, nutrients and micro-organisms the tree needs as well as (some) oxygen for root cell respiration, and ideally should not be disrupted so much so that exudates and microbes (fungi or bacteria) are lost. The risk associated with bare-rooting a tree (or excessive repotting) is that it destroys the rhizosphere all at once, leaving the tree vulnerable to pathogens and forcing it to regenerate exudates it has already created (which can use up to 40% of its stored carbon).

Your tree’s roots need to have a regular supply of nutrients, so they require fertiliser of some kind. Even if good compost is added during potting, the small size of bonsai pots will mean the nutrients won’t stay in there for very long. Trees will need added fertiliser – either home-made (for example, regular doses of diluted compost leachate), or purchased. And obviously – watering is critical. Given the role of symbiotic partners (such as fungi & bacteria), you can also add these to the soil – if your tree senses their presence and wants them to stick around – it will probably produce exudates to achieve this.

The Microbiome and Symbiotic Microbes

It has been known for over a century that tree roots are colonised with microbes, particularly fungi, but it’s only in the last twenty-five years or so that this idea has captured the public imagination, with Suzanne Simard’s discovery that trees can actually communicate and share resources via their fungal networks.^ref

Of course, our knowledge about microbes – a collective name which refers to any living thing so small that a microscope is needed to see it – has massively increased in recent years. Studies into the human microbiome have shown that our own cells are outnumbered ten to one by the cells of microorganisms which live in and on us (Collen). These are mostly bacteria but also include viruses, fungi and archaea, and some of them perform important roles in human health – for example comprising a key part of our immune system.

The same concept applies to trees. Microbes are everywhere on and even in trees, above-ground and below-ground, and some of these are beneficial to the tree, whilst others are detrimental. Microbes colonize the germinating seed right at the beginning of the tree’s life, then move on to colonize the radicle (root) as it emerges and then the cotyledons (first true leaves). Over the tree’s life the species and number of microbes will shift and change. It has been shown in a recent pre-publish study that 95% of the fungi and bacteria present in acorns were transmitted to seedlings, and it is expected that further research will show this is inherited from the parent tree.^ref

So not only do seeds inherit their genes from their parents, they also inherit their microbiome.

The microbiome (community of microbes) of trees comprises the phyllosphere (microbes in the foliage), rhizosphere (microbes in the roots), and the endosphere (microbes within the plant itself). Within these live a wide variety of bacteria and fungi, co-habiting, interacting, supporting and competing, with a range of different impacts to their host. A newly emerging term in this field is the ‘holobiont’ – this is a host with its microbiota and recognises that they interact with each other as well as the host. A tree and its microbiome are a holobiont.

https://neutrog.com.au/2020/04/23/the-plant-microbiome/

To understand more about the microbes in each sphere and what they do, read the three posts I linked to in the previous paragraph, each has guidance relevant to their different domains.

From a bonsai point of view, we want to help our trees cultivate a healthy community of beneficial microbes in their microbiome, since this helps them access nutrients, fight pathogens and stress and thrive. There are three things we can do to help with this. The first is to avoid killing the microbes! For example, adding pesticides, chemicals, anti-biotics, weed-killers, anti-fungals etc could damage your mycorrhizal and bacterial communities. There are hundreds of studies showing that glyphosate kills off AMs and ECMs, and it has been shown to negatively influence microbial survival directly as it inhibits an enzyme of the ‘shikimate’ pathway, which produces essential amino acids in both plants and the majority of microbes.

The second thing is that you can add mycorrhiza and beneficial bacteria to your bonsai soil, particularly if you are repotting and losing the existing communities, also if you are creating new bonsai through collection, seed growing, air layering etc. You can buy dried mycorrhiza and bacteria mixes which can be sprinkled into the pot and watered in – I have my mycorrhiza in a salt shaker and my bacterial inoculant in a pepper shaker. The research is a bit mixed about how effective this is since microbes don’t necessarily establish the required density to contribute to plant defences & health, but you can optimise their chances by ensuring your substrate has plenty of nooks & crannies for bacteria to live (eg. this is one of the main claims for the benefits of biochar). Check the product you are buying to ensure it matches the type of mycorrhiza your tree associates with (some products have both ECM and AM). Alternatively, if you can find some soil or humus from an unfertilized, chemical-free forest with similar species, grabbing a handful and stirring it into your bonsai soil will also add benefical microbes .

The third thing that can be done is to create an environment for your trees which microbes prefer. Good soil, a good level of moisture, drainage, a carbon source (in most cases – roots) and not too much disruption of the roots, good lighting and avoiding large temperature variations, and air flow around the foliage.

Microbes aren’t all sweetness & light though, some are pathogenic not just to plants but to humans as well. Improperly composted manure can introduce bacteria including Salmonella, E. coli and Enterococcus. More relevant to bonsai enthusiasts is the fact that the Legionella bacteria which causes Legionnaire’s disease (a potentially fatal pneumonia) is present in many composts including those made from wood, bark, green waste and peat.^ref As a result, whilst we certainly should appreciate our friendly microbes for their role in our bonsai practice, we should also make sure to wash hands and tools thoroughly, and avoid breathing in any organic matter such as compost. When mixing bonsai substrate, doing this under a cover, outside or in a bag is preferable to doing it in a way which sends dust particles into the air.