International research on root physiology and function – a report on a study tour to European research institutions

Call to action/take home messages

Plant roots are the interface between the soil and the growing crop, so understanding how roots respond to nutrient and moisture availability and other soil characteristics (compacted layers, air gaps) is an important factor in designing more effective management systems.

This trip was funded by GRDC as part of a research excellence award made in 2016, and was undertaken to discover new approaches to the study of root growth and function, and to explore opportunities to develop international collaborations. I visited a number of important research groups in the UK and Europe that are using innovative techniques and equipment to better understand root growth and function.

The main focus seems to be screening genotypes for narrow root angle and hopefully, improved access to deep profile water, but most work is only being conducted in glasshouse pot studies. We are interested in any potential trade-offs between the ability to forage for stratified/banded phosphorus fertiliser in relatively shallow profile layers and root systems that are effective at accessing deep profile moisture.

Background

Productivity of grain systems in the northern region has been governed by efficient rainfall capture, and the storage and extraction of water from the soil profile. Agronomic practices have been developed to improve synchronization of crop water use with key yield-determining growth stages – for example, early season canopy management and wide/skip row planting. Additionally, genotypes of both winter and summer cereals are being screened for traits such as stay-green and root traits that may confer better access to deep soil water.

While these developments can deliver improved crop water-use efficiency, they will only do so if roots are able to function efficiently in the soil profile. Restrictions due to factors such as the presence of subsoil constraints, poorer soil structure, or compaction, may limit the productivity benefits that can be obtained.

Similarly, plants require nutrients as well as water to grow, so root systems that are able to access more water must also be able to access the nutrient needed to support any additional growth. A history of under-fertilization has depleted native soil fertility, and without fertilisers, nutrient constraints would prevent attainment of water-limited yield potentials. However it is sometimes difficult to develop fertiliser application strategies that will deliver a nutrient distribution pattern that matches that of available water. Mobile elements like nitrogen (N) and sulphur will redistribute (slowly) with water during soil profile recharge, so ensuring that nutrients and water are present in the same part of the soil profile is achievable.

For immobile elements like phosphorus (P) and potassium (K), reserves are becoming increasingly concentrated in surface soil layers, or in nutrient-rich bands at the base of the cultivated layer (i.e. 15-25 cm). This stratification is causing nutrient and water reserves to become spatially separated in all but the wettest years, and the response to this by crop root systems will determine productivity in any set of seasonal conditions.

The research programs exploring responses to deep placed P and K fertiliser bands are showing that appropriate placement of P and K fertilisers can provide productivity and profitability increases, but the interaction between seasonal moisture availability, root system structure and activity and nutrient distribution, will ultimately determine the efficiency of these fertiliser application practices.

Techniques to effectively measure root distribution and activity are improving rapidly, and before moving into more root-related research, it is important to understand what works and what doesn’t. To that end, this study trip was undertaken to examine root research programs in a number of leading groups in Europe and the UK. The intent was to determine useful research approaches that could be adopted here, look to develop collaborative links where appropriate and also derive any useful insights for our own research programs.

To that end, a trip to visit leading groups in France (INRA, in Montpellier) Switzerland (ETH in Zurich) Germany (IBG-2 in Jülich), Scotland (the Hutton Institute in Dundee) and England (the University of Nottingham, Sutton Bonnington campus) was undertaken in August-September 2017. Some key learnings and points of interest are reported below.

Techniques for studying root growth and function

Imaging techniques

There were 3 main root imaging approaches that were used to characterize crop root systems.

1. Rhizoboxes – these are relatively simple thin, soil-filled rectangular boxes with 1 face of clear glass, with light excluded to mimic underground conditions. The box is placed at an angle to the vertical, plants are grown in the soil volume and cameras are used to document root growth measured at the glass interface, or after destructive harvests, by washing the soil away from roots suspended on pins or pegs to hold the roots in position. These are widely used for short duration assays of root growth and response to different conditions, but can only be used with loosely packed ‘artificial’ media and quantitative root assessments are labour intensive. There are various degrees of automation in the imaging systems to record root development (see figure 1).
2. X-ray computer tomography (CT) systems - CT employs an X-ray beam passing through the sample which absorbs (attenuates) part of it, thereby reducing the intensity of the beam. X-ray attenuation is mainly determined by material properties, with the internal structure of a sample becoming visible by contrast according to density and atomic number of the elements. A major problem is the often very similar attenuation of roots and some structures in the soil such as water filled pores, making segmentation of the roots (especially fine roots) from the soil background slow and dependent on sophisticated software tools for data analysis. Other problems include difficulty in differentiating between live and dead roots, making analysis of soil cores from the field problematic, with most results obtained for plants grown in small diameter pots with root/residue free media, and the cost of the instruments ($AUD 0.5-1.2M, depending on size). That said, the images that can be produced provide detailed insights of 3D root growth, and also 3D information on soil structural characteristics (see figure 2).
3. Magnetic resonance imaging (MRI) - MRI is based on the magnetic moment of atomic nuclei like ¹H (protons) which are highly abundant in living tissues, particularly in water molecules. The magnetic moment can be manipulated using strong magnetic fields to produce 3D images of samples, although for high quality images, a substrate with low ferro-magnetic particle content is required. A range of contrast parameters can be exploited to highlight differences within a sample, and this can be exploited to produce a strong difference between ‘root water signal’ and ‘soil water signal’ which provides a very high contrast between roots and soil background, overcoming one of the limitations of the CT systems. Water mobility in roots and soil has been shown to be detectable with MRI.
   Constraints again are focussed around the sample volume that can be analysed without losing image resolution. Root growth is typically assessed from narrow diameter (a maximum of 15-20cm) pots or soil cores collected from the field, with the length of imaging time increasing with sample volume. Data analysis is however generally much quicker than the X-ray CT. This is due to the stronger contrasts and the lack of need for detailed imaging software. Price is again a significant constraint (AUD $1-1.3M to build the unit at Jülich, and a team of nuclear physicists to operate it), but the quality of imagery that can be obtained is excellent (see figure 3).

Other systems included a variation of the rhizobox technique without soil, using paper-based and hydroponic/aeroponic systems, and in the field, the development of cheap, flatbed scanners sealed in a waterproof box with a single USB cable to allow downloading of images as roots intersect with the scanner surface during a growing season.

Root function and rhizosphere modification

There have been three main techniques used to quantify the functionality of crop root systems and their impact on surrounding soil in the rhizosphere. These are –

1. Application of tracers – the group using these techniques most extensively were the agroforestry group at INRA, working with eucalypt and mixed sward plantings in Brazil and Mauritius and in walnut-wheat alley cropping in France. The group are making extensive use of Rb (for K), Cs (for Ca) and ¹⁵N tracers to detect root activity and zones of nutrient uptake. Injection of tracer ‘patches’ at different profile depths, along with quantification of root densities and root morphology in those layers through traditional sample collection, has allowed leaching losses of N to be quantified, and demonstrated specialized roots active in the acquisition of different nutrients in different soil layers. Unfortunately the group have also not been able to identify a suitable tracer/surrogate for P uptake, although they suggested we should look at Vanadium in addition to the work we are doing with Mo.
2. Sensing nutrient and pH modification in the rhizosphere – this approach is using pH and nutrient sensitive optodes as a way of quantifying changes in the rhizospheres. All utilize physical interception of growing roots on a treated surface (much like the rhizotrons mentioned earlier), and so are typically used for short duration assessments in glasshouse or field (with considerable installation effort) studies. The acidification of rhizospheres around sparingly soluble Ca-phosphates is an area of interest for our alkaline Vertosols (e.g. see figure 4), as well as for the accelerated weathering of soil minerals and the release of bioavailable K. The latter seems to be particularly important in deep profile acquisition of K by eucalypts in Brazil).
3. Monitoring changes in rhizosphere organisms - assessment of the microbial communities in rhizospheres at different layers is being undertaken using a variety of techniques including substrate utilization (like Eco-Biolog) as well as molecular finger-printing. An area of interest is the potential use of rhizosphere microbial communities as an indicator of how the function of different roots and parts of roots changes with age, distance from the root tip and other factors. Another interest is in decline in root function as roots senesce – will microbes be a sensitive indicator of when roots cease to function in response to a pathogen, waterlogging event or drought?

The main focus of roots research groups

A common theme of the groups where extensive genetic screening of cereal crops was being undertaken, was in identifying variation in seminal root angle, putatively to deliver more effective exploitation of deep soil moisture during dry periods. However, most of this work was being undertaken in controlled environment or glasshouse facilities, and there was not a lot of evidence of evaluating the impacts of that selection on profile water extraction in field studies. This may be a function of the early stages of the research in Europe and the UK (cf. the US and Australia), but also reflected the less well developed links between agricultural research and research funding and the need to find practical solutions to industry problems.

There was also a significant research component exploring root responses to compaction/hard setting (or to pores in otherwise compacted soils), and identifying any potentially useful root traits that could either be used in phases of a crop rotation, or in the target species itself. This focus was probably the result of the continued reliance on conventional tillage systems and the often wet soil conditions under which tillage is undertaken. Like the work with root angle, there was again limited evaluation of potential traits or genotypes in field situations.

There was little focus on nutrient acquisition, with the exception of the INRA group working on nutrient uptake in eucalypt and mixed sward agroforestry plantations, and none of this was focussed on fertilisers. There did seem to be emerging interest in Germany, but that was more related to an impending EU cap on fertiliser use supposed to be enacted in 2020. The focus there was more on the potential to use banded organic wastes as a way of getting round these theoretical constraints to raising productivity.

In all groups visited, there was a strong emphasis on ‘discovery’ science. Sometimes this was related to specific industries or challenges (eg. understanding nutrient and water dynamics in the rapidly expanding agroforestry industry in places like Brazil, or how roots would respond to bands of recycled organic wastes in soils with otherwise adequate nutrient availability), but often simply for the development of new science and measurement techniques. It was often difficult to gauge the potential impacts of some of the discoveries on future productivity or sustainability. There was real interest in the Australian funding model as a way of developing closer links to industry issues in future.

Other observations and interesting research

An immediately obvious contrast to Australian cropping systems was the almost exclusive reliance on tillage. The combination of small fields and wet and often cold soils at the start of the season clearly contributed to this, as did the regular use of cover crops after harvest of the primary crop in late summer/early autumn. However another contributing factor (particularly in Scotland and Germany) appeared to be a desire to avoid use of herbicides/agricultural chemicals. There were a number of references to no till systems only ever being useful for a couple of seasons, due to developing weed pressure, but increased use of herbicides was not considered a viable option to change this. The extent to which this applied to other cropping areas in Europe is not clear, as improved fallow moisture storage was not an obvious priority in these wetter areas.

The widespread use of cover cropping and production of silage (from maize) were also very obvious. It would appear that the popularity of cover cropping was quite clearly linked to the payment of environmental subsidies by the EU for adopting environmentally friendly practices, of which fallow cover cropping was deemed one. From what I could glean, the intent was to minimize the chance of leaching losses of nitrate-N during autumn and winter, especially in areas where there was limited snow cover but wet weather. While the whole crop removal with silage production may reduce the risks of nutrient loss in this fashion, the high fertiliser rates used would still ensure some risks.

There were undoubtedly other benefits from cover cropping, particularly in terms of organic matter inputs to counter the losses from conventional tillage and the introduction of species diversity in systems often dominated by wheat or barley, but it will be interesting to observe the fate of cover cropping in the longer term.

The looming Brexit enactment has a number of other implications for agriculture in the UK, particularly in terms of economic returns. Environmental subsidies (such as for cover cropping where the grower gains no crop return in otherwise fallow periods) can represent a significant component of farm income, and in some cases may mean the difference between viability or otherwise of small farming ventures in more marginal areas. If Brexit results in cessation of such subsidies in the UK, the economic fallout for farming and rural communities could be significant.

The final interesting observation was around concerns for future fertiliser inputs in the EU. The proposed capping of fertiliser usage at current rates for future crop industries is raising concern that this may ultimately put a brake on productivity increases. This has caused a renewed interest in beneficial use of recycled organic wastes, including animal manures and also by-products from bioenergy crops, with a focus on subsurface banding to ensure maximal benefits. This may provide some useful opportunities to link with subsoil manuring/amendment research in Australia.

Opportunities for international collaboration

The leading research groups have taken the study of root systems to another level, with IGB-2 at Jülich and the group at University of Nottingham (Sutton Bonnington campus) in particular having established superb facilities with a critical mass of staff and students providing very dynamic and productive working environments. The strength of these institutions and the excellent facilities they have has helped to foster the development of collaborative links with smaller groups in the region around particular issues or research projects (e.g. the Hutton Institute with Nottingham), and there is genuine interest in developing links to institutions in Australia where interests are complimentary.

The IBG-2 group already have a Science Bridge project with the University of Melbourne, so there are prototypes in place. Both institutions see such linkages giving their researchers and students exposure to real industry problems and field (rather than glasshouse) research approaches, whereas for us the links to state of the art facilities to explore specific research questions would clearly be advantageous.

Where to from here?

The visit has given a different perspective on a number of issues associated with the interaction between root systems, the soil matrix and applied fertilisers. An immediate example would be the impact of application method on the likelihood of recovery of deep P by crop roots, given the observed impact of voids in the soil profile on root proliferation. The latter is the key to good fertiliser P uptake, but if the soil has not reconsolidated around the fertiliser band and the fertiliser is often sitting in air gaps/voids created by the tillage operation, crop recovery will potentially be poor. If this theory is shown to hold true, then there may well be implications for the timing of deep P applications (as early as possible in the fallow), and the type of tillage equipment and subsequent tilth left after application.

In the longer term, it is clear that there is limited understanding of plant responses to dislocated sources of water and nutrients – a situation that we are increasingly finding in northern cropping soils, but which is generally very rare in Europe and the US. There are a number of aspects to this:

• Will P and K-depleted subsoils result in shallower root systems less effective at exploiting stored profile water?
• Will strongly stratified P profiles limit the impact of genetic selection for narrow root angle/deep rooting traits on crop water extraction and yields?
• How will root responses to banded organic amendments (manures, recycled organic wastes) differ between soils with high (Europe) and low (Australia) background nutrient availability?

Finally, it is obvious that there are not necessarily well developed linkages between the study of root traits and function under controlled environment/glasshouse conditions and the performance of root systems and crops grown in the field. The developing ability to collect large intact cores from the field and work with them to grow plants in ‘realistic’ soil profiles for a whole growing season offers a way of addressing this. These cores can be relocated to controlled environment facilities, where manipulation of moisture, temperature or nutrient supply in parts of the ‘profile’ can be implemented.

There was considerable interest from researchers at both Jülich and Nottingham about opportunities to complement their controlled environment/MRI and X-ray CT studies under controlled conditions with realistic assessments of root responses in ‘real’ field profiles, and this interest could be readily developed into a focussed but well-grounded international collaborative research effort.

While the ultimate test of the value of different root traits in the field lies in the impact those traits have on root growth and function, and through that crop performance, the actual quantification of root growth remains hard work. Tools that allow the monitoring of root functions (e.g. monitoring water and nutrient extraction, rhizosphere modification or the microbial communities present) offer opportunities to overcome this limitation and provide guidance in the development of practical outcomes for growers and industry. The development of such tools will be an important part of future roots research programs.

Figure 1. Sequence of rhizoboxes in the glasshouse at Julich. Boxes are on an automated conveyer system that delivers each box to the camera at a defined interval for image collection.

Figure 2. Using X-ray CT to determine soil structure in soils from tilled and untilled fields. False colouring has been used to differentiate soil and air-filled pores (A-C), and soil, water and air distributions (D, E). Image reproduced from Morris et al. (2017).

Figure 3. An MRI image of the root system of a bean plantgrown in a 30cm tall pot with an internal diameter of 8.1cm. Image reproduced from Metzner et al. 2015.

Figure 4. Image of the acidification pattern developing around roots using pH optodes (reproduced from Blossfield and Gansert 2007).

Acknowledgements

This study trip was undertaken by the author thanks to the support of the GRDC, as part of the 2016 Research Excellence Award. The author would like to thank GRDC for their continued support, and acknowledge the generosity from hosts at INRA in Montpellier, ETH in Zurich, IBG2 in Jülich, the Hutton Institute in Dundee and the University of Nottingham (Sutton Bonnington).

Contact details

Prof Mike Bell
University of Queensland
Building 8117A, Gatton Campus, 4343
(07) 5460 1140
m.bell4@uq.edu.au