Microbial response to extreme climatic events

What is root scanning?

(And why does it take so long?!)

Our lab has been using the root scanner as a tool for root trait analysis for a few years now. The scans are analysed with WinRHIZO software which provides (among other things) structural root trait information such as total root length, average root diameter and root volume. As in this paper by Alex Williams.

In a previous experiment, I scanned a great many samples comprised of a single plant still joined at the root crown. This made the cleaning process easier as you had something to hold on to while washing all other material away. Our current experiment, which is investigating community effects, has 12 plants in each pot. For this reason, we chose to take three soil cores instead of root washing the whole pot.

These three cores meant we had less soil to wash away, and less root material in total, but it also meant that we generally had no anchor points. All of our roots are free agents, and vary greatly in size. Root washing our cores without losing any root material proved to be a tricky task. We used a system of sieves, beakers to dip, and medium power spray head. These samples were then stored in ethanol over Christmas.

In January, the laborious task of the final picking and scanning began. This is the really up close and personal cleaning process. Each sample is rinsed then a section at a time is picked over with tweezers to remove any remaining bits of grit, above-ground material, moss or clearly already dead plant material (because we want a snapshot of the root system at the moment of harvest, not any decaying material from before).

This is done in a series of takeaway style or transparent tupperware tubs in front of our large lab windows, to take advantage of the light. Many of my colleagues pity me for having my nose in a tub of rooty water all day most days, but luckily I find it quite zen. There’s a sense of satisfaction hunting out the bits of debris that shouldn’t be there.

The amount and type of root, and amount of unwanted material varies greatly between samples, as does the time they each take but it is common for one sample to take many hours. I have found small paintbrushes can be helpful in catching moss fragments, and have made a range of tiny sieves out of the plastic mesh we use to make litter bags, to aid in catching the roots we want.

Once the sample is satisfactorily clean, it is gently poured into a perspex tray on the scanner bed. Additional DI water is added until the roots are covered. Only ever using plastic tools, the roots are carefully teased apart and spread out until they form a layer one root deep (or as close as we can get it). The scan itself usually takes about five minutes. In that time, we prepare a paper bag, blue roll to pat the sample dry, and a sieve to catch it when we drain the DI water. We record the fresh weight then place the sample in an oven to dry.

Experiment Three Harvest Summary

The last few months have been very busy for our project. Added to the general chaos of life in an everchanging pandemic, we haven’t found the time to update our social media very much. We have been productive though; the greenhouse phase of experiment three was completed before Christmas so all efforts are now on the lab work and analysis.

The greenhouse phase of experiment three was comprised of an acclimation period, where all pots were watered by weight. Then we started a staggered disturbance period, where a third of the batch was subjected to drought and a third subjected to flooding with the remaining pots being our control sample. Half of our pots were harvested immediately after the disturbance period. The other half were given a recovery period before the staggered harvesting continued.

What do I mean by “harvest”?

Pots are weighed and watered.
IRGA measurements were taken from intact pots, first in a light chamber then in a dark (blacked out) chamber.
The above ground material is cut at soil level and placed in a separate paper bag for each individual plant before weighing.
Three soil cores are taken from each pot and pooled in one bag.
The remaining soil is sieved and collected.
This soil is then subsampled into falcon tubes ready for KCl and water extractions. Subsamples are also taken for DNA analysis.
All of this material is then moved from our botanical grounds to the laboratory.
The above ground material is put in an oven to obtain the dry weight.
Soil from each sample is weighed into a tin to be dried at 105°C.

After the harvests were complete, we set to work on the extractions, extract analysis and root washing. Root washing was the longest task and was particularly fiddly because we were washing cores not intact root systems. We used various sieves, beakers, tweezers and our purpose-built root washing station to separate the roots from the soil, and to get rid of as much of the grit, moss and decaying old root material as possible. The next stage for these samples is to go through a final tweezing before being scanned which will continue for the next few months.

The New Greenhouse and the Next Phase

On my first day back at work after a short holiday, and Océane’s first day out of quarantine after flying back to Manchester from France, something wonderful happened… The university took official ownership of our shiny new modern greenhouse building! After years of general delays and 6 months of COVID delays, we were finally allowed to move our experiment into the climate controlled greenhouse.

First we had to put together our new benches though; as I say, we were literally the first people to move in. While the building is now complete and safe, it’s still waiting for some furniture, fittings and finishing touches. Unable to find the proper tools on site, building the tables took a few hours. We used the one functioning rickety trolley to move all 200 pots across from the polytunnel. It was a surprisingly physical day (each pot weighs around 2kg) but massively satisfying and I must admit I allowed myself a celebratory box of chocolates that evening! After being solely responsible for keeping the plants alive and healthy, and making all the practical decisions for the experiment for six months, it was a huge relief to get them into the greenhouse and to be working as a team again.

Now able to properly control the climate and with Océane back in the country, we started the acclimation period. After two weeks of controlled temperature and watering by weight, the disturbance period for batch one will begin.

We started the acclimation period immediately, taking moisture measurements from all of the pots to assess the baseline and calculate what weight to water to.

Experiment 3 Progress

We are now about 20 weeks into the COVID-19 pandemic in the UK. The lockdown was largely lifted across the country but then a local lockdown was reinstated in Manchester. Thankfully the new rules do not affect our access or ability to proceed.

The University of Manchester started slowly reopening research facilities in phases about a month ago. Some of our lab members have already gone back to work, following strict new health and safety guidelines. Our group has also set up a scheduling system to ensure we follow the rules, don’t exceed maximum occupancy, and people get fair access to in demand instruments. Océane and I have recently been added to the access list, in preparation for the labwork associated with our two harvests.

Back at the polytunnel, our pest measures were fairly successfully in controlling the aphid issue. Our plants are now a little older than we initially planned so we are giving some thought to how best to proceed. We have had both extremes of weather to deal with over the last few months, from thunder storms to heatwaves. Even with the industrial fans on, repeated watering and all the doors open, the polytunnel reached some uncomfortable temperatures in the high 30’s! I researched some cooling “hacks” online and tried an ice tray in front of the fan, inline or adjacent fans and wetting the surrounding ground (with unknown results).

We also used the Ikea click and collect service to purchase some plastic plates to use as pot saucers. We were surprised to find a complete lack of the correct size saucers available online in the time frame we needed, but then, this was at the height of the lockdown. The saucers were essential as they allowed us to water from the bottom. This was vital not because of the heat (although that later proved useful) but because our pest control critters don’t thrive under heavy watering.

I have also planted out a batch of new pots, which still have 12 individual plants but of all the same species. The idea of these single species pots is to use them for root trait analysis to accompany the main experiment.

After years of promises, the university finally started to replace our old glasshouses late last year. Only the skin of the new building was up when the pandemic hit but the builders did continue to work during lockdown, albeit in reduced numbers. After months of slow progress, we have been assured that the inside is now pretty much complete. However, we don’t know how long the snagging list and hand over process will take. Ideally, we would start the treatment phase of our experiment in the new greenhouses which have more control and better facilities than the polytunnel.

Ladybird larvae in lockdown

By the time I finished the first pass of weeding, the pots at the beginning already had some new weeds sprouting, as shown in this photo of a pot that should be a soil blank.

I ordered some fly paper when I first noticed the aphid issue – it isn’t particularly helpful against the ones already on our plants but it may help stop the spread to other pots, while we are waiting for the proper solution to arrive. It’s not possible to suspend things from the roof of the polytunnel so I dug some canes out of the dusty old potting shed, extended them with support sticks which slotted neatly into the natural hole in the bamboo, and poked them through the holes in the table. The bright yellow of the flypaper creates a striking image!

Then our secret army of helpers arrived in an unassuming cardboard box. We had to extricate our ladybird larvae from among the seed cases they were shipped in. It was a fiddly job, made worse by how fast and agile the larvae were! I used a soft paint brush to gently move them around and started putting one in every pot, and two on the pots with the worst aphid issue. But it became clear that we didn’t have as many as I had hoped and I changed to putting them only in pots with visible aphids. This took place in the first week of the unseasonably warm weather, which presented a few issue for us. Firstly, the polytunnel has no climate control mechanism so it warms up very quickly when the sun shines meaning the pots require more water. Secondly, the ladybird larvae don’t like rain, so watering with the high power sprinkler hose was out. These factors combined led to me, wearing as little clothing as is decent, watering each pot by hand with a watering can, trying to avoid splashing water on the leaves.

The heatwave continued for weeks, which may have been enjoyable for the furloughed staff with gardens, or the kids taking advantage of local parks, but it was a disaster for our anti-aphid plan. We recorded a temperature of 40°C at one point, and even with all three doors open, and the heavy duty fan on full power, the polytunnel was often well over 25°C. While there was an initial improvement in aphid numbers, either the heat, the extra watering or some combination of both seems to have caused the ladybird larvae to leave or die. Two weeks after application, I couldn’t see a single larva in any of the pots.

The aphids, on the other hand, continue to thrive. On my second weeding pass, I carried on picking or wiping off aphids where possible. It does not feel like an efficient solution though, so we may try another batch of ladybird larvae, this time avoiding a heatwave and watering the pots from the bottom. For this to work we need to find and purchase saucers for each of our pots, which are big enough for us to easily pour water into, but not so big that they no longer fit on the finite polytunnel bench space.

I managed to free up some more bench area by cleaning up an experiment that had ended, so was able to space our pots out more. This will hopefully help with aphid spread and air flow. I am also planning to rotate our blocks soon, as some areas of the polytunnel get more sun than others.

The new greenhouse building are supposedly close to completion, but no one seems sure when we will actually be able to start using them. I know the Covid-19 and furlough situation is making it hard to get contractors in to finish up the inside. Many of our plants are quite substantial now. In fact, the pots containing the fastest growing species can be quite difficult to water and weed, and impossible to de-aphid as the plants have grown into a tangled mess, spilling over the sides or swamping the whole pot. Uncertainty is ubiquitous at the moment, so we continue to cross our fingers and hope for the best – what else is there to do?

Polytunnel Progress

The University of Manchester is still in lockdown, with all learning online, and every member of staff possible working from home.

So for now, instead of the swanky new greenhouses we were promised many years ago, we are growing our plants in a large and slightly draughty polytunnel – but it’s better than nothing!

Each pot contains 12 individuals, which were planted in a set order following a template. That formal planting scheme will be important when we come to harvest and is already proving useful to distinguish between our desired plants and unwanted weeds.

Chemical weed killer isn’t an option, as it would effect the soil and our plants, so we have to pull each weed out by hand. It’s a simple if time consuming job, but at the moment it is just nice to be out of the house!

Unfortunately, I also discovered this week that a neighbouring experiment has an aphid infestation. Those plants were established enough to be moved outside, and I gave the bench top a really good clean but I will have to keep an eye out over the next few weeks. Again, chemical pesticides are not an option, so the plan of attack is fly paper and more tweezing!

Corona Chaos

Like everyone else, the COVID-19 coronavirus pandemic, social distancing measures and subsequent lockdown in the UK has had a huge impact on experiment three of the project.

After months of preparation and methodical batch germination, we had just reached the potting out stage of our microbial community experiment. The plan was to plant our seedlings in four batches with a week between each, to give ourselves time to harvest such a large experiment. But the week I wanted to plant batch one, all hell broke lose in the UK. With Coronavirus cases on the rise, the university of Manchester made the decision to close all university buildings. There was a lot of confusion and uncertainty for the first week; a lot of back and forth on who should be allowed what access, while government advice changed regularly. Eventually they decided to impose the same strict restrictions on the botany grounds as the main buildings which was very unfortunate for us.

We held a zoom meeting to discuss our options, where (after briefly considered buying cheap greenhouses for my personal garden), we decided the only course of action was to scrap the staggered batch planting and watering by weight during the growth period. Which meant we had from midday Friday until Sunday night to get as many pots planted as possible. It would have taken at least 8 days for me on my own, but thankfully some amazing lab colleagues donated some time across the weekend. We tried to maintain a precautionary distance from each other as much as possible, even though this was before the strict lockdown was announced.

To speed up the process, we would pick out enough individual plants of each species for one batch at a time and keep them moist between damp blue roll. A template was used to mark out where each plant would go, and they were planted following the same pattern each time to alternate grass and herb, and maximise the distance between the two individuals of the same species.

It was a very long weekend, in fact I even took some trays of plants home each night to pick out individuals for the next day. I also had to do some of the planting by head torch, but it was worth it in the end, as we managed to get three out of the four batches planted. Of course it’s disappointing that we can’t execute the experiment as we originally planned, but at least we have something growing for the next few months. This was an incredible relief, as it had looked like we might lose the whole experiment at times.

One member of senior staff is still allowed access to the botany grounds and has agreed to water our pots and send me the odd photo of their progress. We don’t know how long the university buildings will be shut but we obviously cannot start the treatment phase until we have access again. The original plan was to let them grow for 8 weeks, but a little longer should be fine.

It’s a time of unprecedented uncertainty and chaos for everyone in the country, so like many people in science, for now we just have to wait and see what happens.

Thank you to Chris Knight (@knightjjar), Danna Gifford (@dannagifford), Chris Sweeney (@ChriSweeney) and Hayley Craig (@hayzleypop) for their invaluable help.

Introducing myself

Hi,

I am Océane, I arrived in the Soil Ecosystem lab 2 years ago to work on this project as a postdoc. I did my PhD thesis on plant/microbe interactions in a small city called Nancy, located in the North East of France.

I studied a beech tree forest, where, despite a natural pH and nutrient gradient, tree productivity was surprisingly even across the site. While studies evidencing the role of microorganisms in tree health and productivity were accumulating, I investigated the role of mineral weathering bacteria located in the rhizosphere of beech trees in this process. I found out that beech trees recruited efficient mineral weathering bacteria in their rhizosphere, thus increasing the availability of inorganic nutrients, exclusively in nutrient-poor soils. Meanwhile, no enrichment was observed in the bulk soil. This study permitted us to dig deeper into the mechanisms of recruitment of a specific rhizosphere microbiome by trees, depending on surrounding environmental conditions, in particular those which can induce stress. During this period, I specialized in classic microbiology, made several bacterial collections and tested their functions. I also started studying molecular microbial ecology to unravel the structure of microbial communities in forest soils. This project gave me an insight into the functioning of academia, the publication process, and inspired my interest in R and statistics more generally. Plus, I discovered a new hobby of making nice plots.

Although this period was really enriching for me and I really enjoyed the labwork, fieldwork in the forest and the computing work, I found it a little frustrating to not be able to extrapolate my results to other sites. I ended up realizing that in science, the more we know, the more we can apprehend how much we don’t know. After this experience, I wanted to be part of a broader project, where I could observe microbial communities on a larger scale, on more samples, to be able to draw big trends of microbial distribution. I was also expecting to sharpen my bioinformatic and modelling skills with a new project.

This is how I ended up at the University of Manchester, working on a fascinating project designed by Franciska, with Chris, Carolin, Holly, Rob and Tim, on a subject that really matters to me. Instead of talking about global warming, now most of us know that we should talk about “global change”. We’re not only expecting a rise of average temperatures, but also an increase in the frequency and intensity of extreme climatic events, such as hurricanes, drought, heat waves, flooding or freezing. The past few decades have shed light on the role of soil microorganisms in several soil ecosystem functions, like biochemical cycles, nutrient recycling, and organic matter decomposition. While an annual change in climate could alter microbial communities progressively, extreme climatic events can abruptly disrupt microbial communities, with unknown consequences on their long-term resilience and on ecosystem functioning. The main threat is a loss of soil biodiversity, resulting in a reduced soil multifunctionality and a loss of ecosystem services such as soil fertility and plant productivity. However, predicting soil microbial community response to extreme climatic events is difficult as it depends on several parameters interacting with each other, like soil properties (pH, moisture, nutrient availability), plant coverage (diversity, community composition) and climate history. This project aims to unravel the factors driving soil microbial response to extreme climate events through three experiments, with a focus on a different aspect for each (see figure):

1. Thirty natural grassland sites across on a European gradient, where soil type/climate/plant community vary (many confounding factors but many sites)

2. An experimental site (Raindrop) where climate has been manipulated for four years (rainfall addition/exclusion) but soil type/plant community are constant

3. A mesocosm experiment where plant communities had been manipulated to obtain a gradient of functional traits, where soil type/climate are constant

For those three experiments, the soil samples collected were subjected to several disturbances in the lab (drought/flooding/heating/freezing) to observe the response of soil microbial communities. In particular, the aim was to identify the response and effect traits related to the resistance and resilience of microbial communities.

At this stage, experiments 1 and 2 have been set-up and harvested and I’m modelling the response of soil microbial communities and soil functions. Experiment 3 has recently been set-up just before the shutdown of the country by Holly and we’re currently expecting to harvest in August/September. Hopefully, those experiments will allow us to have a better understanding on the factors driving microbial resistance and resilience to extreme climatic events.

Stay tuned if you want to know more about this project!

Writing Retreat

And what a treat it was!

In early March, the Soil and Ecosystem Ecology Lab headed to the Peak District for a writing retreat. The trip was instigated by Dave Johnson, and planned mostly by Debbie Ashworth, Hayley Craig, Mathilde Chomel and me (Holly Langridge). The majority of the lab, from MSc students to post-docs, descended on Roaches Hall near Leek on a clear Tuesday afternoon.

The first job was to receive a mammoth Tesco delivery – it turns out buying food for 20 odd people for 6 days is a big complicated job! As well as breakfasts and lunches, we had the ingredients for volunteers to cook the evening meals – which were all a culinary delight! As well as cooks, we created a rota of helpers and cleaners. Our handmade pizza on Thursday night needed to prove for a while so we found it a sunny spot on the window sill.

We spent the first evening settling in to the house, ready to start the real work bright and early. We employed a mix of structured and relaxed writing sessions, with one room set aside as a quiet space. Everyone wrote down their aims on the first morning to help prioritise and monitor the success of the retreat. I found the lounge was the most comfortable place to work, and was lucky enough to bag the bucket seat by the window, overlooking Ramshaw Rocks.

As well as a spacious dining room and lounge, the house contained a pool room, two X-box nooks, a table football den, a slightly crooked ping pong table and pleasant garden. We made use of all of them after work hours, and pool was a particular favourite. We also brought a duffel bags worth of our own games, which provided great opportunity for fun and team building.

One of my organiser tasks was to plan activities for the mid-afternoon break. All the advice and research online suggests getting your mind off the work is important to productivity. I opted for activities that got people moving; a treasure hunt on the first day and the egg drop game on the second. I wrote and hid the clues on the first night which led to two separate people asking if I was alright, as I loitered in the hall waiting for an opportunity to sneak a piece of paper into a plastic flower!

As well as working on our individual projects, almost everyone gave a short presentation, and a few people split off to work on group projects. My main workload over the week was proofreading two papers and writing up methodologies for my current and previous projects.

We had the house booked over the weekend too, but the writing was done. So on Saturday we could do whatever we wanted. Most people chose to take advantage of our amazing rural location and headed out for a walk (of varying length). I joined the less strenuous of the two walks, which extended from our front door over the Roaches and to Lud’s church. We were fairly lucky with the weather, which was clear for most the day and never rained although the wind was exhilarating on the way back. Later on Saturday evening we headed to a local pub for a meal no one had to cook.

On Sunday, most of us packed up early and headed back to Manchester. Some people (and their partners) hung back to enjoy the space a little longer.

I can’t speak for everyone else, but the retreat and lack of distractions it provided, proved very productive and enjoyable for me. I’d definitely recommend the premise for other groups – although location choice is key!

Suboptimal Conditions

I’ve been sieving the soil (not constantly you’ll be pleased to hear) for about two weeks now. Big, dirty jobs like this generally take place at the university botanical grounds, colloquially known as The Firs. We used to have a row of large but very dilapidated glasshouses. They were torn down last year so a modern replacement can be built but as usual with building work, it has taken longer than expected.

We collected over 750 kg of soil from Lancaster in late December but it needs sieving to remove debris/roots and to help homogenise it. Sieving is extremely difficult if the soil is wet and has a large clay content so we sliced the bags and laid them out over the Christmas holiday to dry. Most were still too damp when it came time to sieve so I set up a couple of fan heaters in the potting shed and have spent the last three weeks turning the soil and rotating bags.

Currently we only have access to a drafty poly tunnel or a practically Victorian potting shed, which is where I set up. Unfortunately, and completely inexplicably, the power system in the potting shed seems to hate me. When I first started sieving, we could have all of the lights on, with both heaters turned up. Then the heaters started tripping the fuse unless we turned them right down or used only one. I managed to keep one heater on medium and charge my tablet on one day, but then that became too much. All leading to today, when I finally had to choose between having the light on or one heater on low. Given that it was daylight but freezing cold, I opted for the heater!

It’s not all bad working at the Firs though. The high walls keep it quite separate from the buzzing student area that surrounds it. With the wildlife pond, open grass and exotic glasshouses it’s actually a peaceful haven when the builders aren’t in. Sieving is a fairly monotonous job so along with listening to podcasts I’ve watched quite a few episodes of Bones in the last two weeks. It’s my second time watching the series so it doesn’t matter that I spend half of my time looking down at my hands to untangle the roots or pick out the stones.

While endlessly standing at the potting shed work bench, turning the sieve crank, I’ve also had a front row seat for a lot of the building work taking place at the moment. Including an amusing 15 minutes saga while they tried to drive two small scissor lifts through a set of doors only about 10 centimetres wider than the machinery. The drizzle, loose temporary path surface and ramp leading up to the doors (at an unhelpfully acute angle) made for a comedy of errors. I witnessed many attempts, driver changes, skidding, shouting, group manhandling, drivers and pushers trying to bounce the lifts, and thankfully a well timed last minute duck as the scissor lift finally found traction and sped through the doorway.

More than half of the soil is done now, which is good as the seeds have been planted and the next big step is to prepare the pots. Progress might slow though if we can’t get some kind of heating and lighting fixed up in the potting shed. Taking warmth breaks in the tropical greenhouse is fun but not productive, and it’s set to rain every ten minutes!