 Good morning, good afternoon, or good evening, depending on where in the world you are tuning in. I'm Max Hegblom, Editor-in-Chief of Femmes Microbiology Ecology, and it is my pleasure to welcome you to this webinar on the ecology of soil microorganisms. Our speakers today span the globe from Europe to North America to New Zealand and looking at who's logged in, we also have a global audience. So thank you for joining us today. Femmes continues the series of webinars to support the microbial community. As a not-for-profit organization, FEMS, the Federation of European Microbiological Societies, uses the income from our journals to fund our charitable activities and support our community. FEMS journals, indeed, invest in science. We provide grants to scientists, organize and support conferences, and sponsor a range of events such as this webinar series. These webinars provide a forum for the presentation and discussion of key research, enabling the flow of ideas despite the cancellation of in-person events and conferences right now. Each month we are highlighting a different topic of microbial ecology, and so if you missed our earlier webinars, they are also available via the FEMS and Oxford University Press websites. Today we focus on the fascinating topic on the ecology of soil microorganisms. Indeed, soil is alive with diverse microbial communities, bacteria, fungi, archaea, protozoa, and viruses, with complex interactions and ecological networks. So, microorganisms are the driving force of biogeochemical cycling and a bridge that links above and below ground ecosystem interactions and key ecosystem processes. Today we have presentations by Michael Van Nuland, Amanda Black, and Fleming Ekeland, who explore three different soil habitats. A temperate boreal forest ecotone, old growth cowery forest in New Zealand, and agricultural and forest soils. And they examine the effects of climate change, loss of indigenous three species, and the effects of soil amendments. After the three talks, we will open the session for questions and discussion, and you can submit your questions via the question link in India GoToWebinar platform. So, again, our three speakers, Michael Van Nuland, Amanda Black, and Fleming Ekeland. Our first speaker, Michael, is a postdoctoral scholar in the Department of Biology at Stanford University, and he will discuss warming and disturbance, how it alters soil microbiome diversity and function in a northern forest ecotone. Michael, the floor is yours. Thank you, Max. Let me share my screen here. Okay. Yeah, so thanks everybody for virtually tuning in. My name is Michael Van Nuland. I'm a postdoc in the biology department at Stanford University, and today I am close. Hang on one second. Okay. I'm trying to close that. Okay, great. Today, I'm excited to share some of my work on how the soil microbiome responds to environmental change in forest ecosystems, and I'm excited about this work because it helps us understand a little bit more about the importance of interconnectivity of microbial communities and determining community responses, as well as thinking about how structure and function are linked when microbial communities respond to environmental change. And I want to quickly acknowledge my co-authors on this work that are spread across the US and the Department of Energy for funding for the experiment that I'll be talking about. So soil microbes are incredibly important regulators of forest health and ecosystem function, and that's because they do important things related to biology or chemistry cycle below ground, as Max was mentioning. Mycorrhizal fungi, for example, form really extensive symbiosis with plant root systems that protect them from herbivores and also help plants access essential resources and nutrients from their environment. Freeliving fungi and bacteria also help mine minerals and nutrients from the environment. And of course, these activities have important consequences for the global carbon cycle. Their activities, they respire CO2 into the atmosphere, but they also help stabilize carbon below ground through the decomposition and turnover of complex organic molecules. And so if we want to understand how soil microbes respond to environmental change, like climatic warming, important areas to look are spots with lots of soil carbon. And here it's important to point out that soil carbon is not evenly distributed across the world, that there is a lot more of it in high latitude forest systems. The map on the left shows how the density of soil organic carbon increases in these high latitude forest systems in the blue and green areas. And the right figure shows how the total soil carbon stock increases with latitude as well. In North America, these high latitude forest systems are dominated by boreal tree species. So this is the dark green area stretching across Canada and parts of the U.S. These boreal forests are typically made up of spruce and fir tree species, for instance, but interestingly, there's this important ecotone or transitional ecosystem between temperate forests that's more southern latitudes that are made up of oaks and maple tree species, for instance, where they coexist with the southern range limit of boreal forest tree species in this sort of lighter green band stretching across North America. And this transitional ecosystem is really an interesting and important area to look at environmental change because the tree species are responding differently. Temporate tree species have been shown to typically respond positively to warming because they're at their colder northern range limit, whereas boreal tree species have shown to respond negatively to warming because they're at their warmer southern range limit. So this ecotone represents a really important and interesting place to look at soil microbial responses to global change factors because you could imagine they're responding in multiple ways, right? They could respond directly by changes in environmental pressures like warming or disturbance. They could be responding indirectly based on differences in tree species performance as the trees themselves are responding to change, or it could be some combination of the two. And so to look at this a little bit further, I took advantage of an existing experiment that sits right in the middle of this ecotone in northern Minnesota in the United States called B4 Warmed or the Boreal Forest Warming at an Ecotone Endangered Experiment set up by researchers and co-authors on this paper at the University of Minnesota. This experiment consists of above and below ground warming in realistic forest plots. So you can see the photos above. These are warming environments outside in natural forest environments. And the warming experiment was set up in two different canopy disturbance treatments, closed canopies and open canopies, which represent important disturbance events in these forest systems that are important for the regeneration of certain tree species. So zooming into one of these plots, for instance, you can see in the bottom left photo is a zoomed in photo of what these plots look like. They have both above and below ground warming, like I mentioned, below ground warming from resistance heating cables and above ground warming with these infrared lamps to create three different levels of warming ambient where the entire setup was generated and the switch just wasn't flipped on about a two degree Celsius and about a three and a half degree Celsius increase in temperature. And like I mentioned, under two disturbance or closed versus open canopy treatments. So we sampled 36 plots, six replicates of the three warming and two canopy treatment design and each of these plots had 121 seedlings planted in it that represent five Boreal, five temperate and one non-native tree species in the system. We sampled five soil cores from these plots that were pooled at the plot level and extracted DNA. We performed our microbial sequencing on a MySeq platform using 16S and ITS2 amplicons. And then I processed the data using the data to bioinformatic pipeline, which generates Amplicon sequence variants or ASVs. This resulted in a little over 3,000 bacterial ASVs. And I used the fungal database for the fungal side with the ITS amplicons to pull out ectomycorrhizal fungi versus saprotrophs. And ectomycorrhizal fungi, just to jog your memory, are the type, one type of mycorrhizal symbiosis with plant roots that sort of form exterior structures and kind of squeeze their way in between plants several roots. And saprotrophs here represent more of the free living decomposers. Now in the paper, I talked about all three of these groups. In this talk, I'm only going to focus on the two fungal groups. One of the reasons is because there's a really strong link between the activity of ectomycorrhizal and saprotrophs in four systems and their extracellular enzyme capabilities to decompose and degrade lignified or highly cellulitic plant material. And we've we performed a soil enzyme assay from the soil cores as well to measure some level of community functional potential. So using this data, I'm going to be talking about three main research questions today. First, how did the different fungal groups that I mentioned respond to the warming and disturbance treatment? So kind of a classic microbial ecology test. The second, does connectivity predict the amount of fungal community change? And I'll talk a little bit more about what I mean by connectivity in a second. And third, are fungal community changes related to shifts in soil enzyme activity? So is structure related to function here? So jumping into this first question and again, focusing on these two fungal groups, looking at Shannon diversity, first of all, across different warming treatments, we found not really a strong response. As you can see, all the all the colors and open versus closed points are kind of clustered together. So we didn't find a strong overall diversity response. We did find a pretty strong compositional ship. So this is a principal component analysis where points closer together represent samples with more similar community composition of ectomycorrhizae in this case. There were strong effects of warming and canopy disturbance on both community composition of ectomycorrhizae. And when we look at how relative abundance of different ectomycorrhizal phyla change, we find a pretty strong decline in vicidio mycota, which include ectomycorrhizal genera like clavalina, quartinarius, lactarius, and tomentella. And ascomycota slightly increased with the canopy disturbance. So we see these sort of different phyla level responses to the two different treatments. And there were no interactive effects at the phyla level here. So showing the same types of plots for saprotrophic fungi, here we do see a slight bump in Shannon diversity with closed versus open canopies. So they seem to increase their diversity slightly from canopy disturbance. Relatedly, we see a stronger compositional shift of saprotrophic fungi with the canopy disturbance versus the warming treatment. So you can see the open kind of convex holes shipped a little bit more to the right in the principal component analysis graph. And at the phyla level, we don't see any strong response at the vicidio mycota or ascomycota level. So suggesting that their community responses are happening at a finer tax non-presolution. One other thing we looked at was the ratio of ectomycorrhizida saprotroph relative abundance in the system, showing that this ratio actually declines with warming. So it's suggesting that the balance between roots symbiosis to free living decomposers in the system is responding to these warming treatments in ways that could have implications for carbon cycling. And we think this this type of ratio shift is happening because password has shown that boreal tree species that might be more reliant on ectomycorrhizofungi are responding negatively to warming. So perhaps the ectomycorrhizae aren't performing as well as their tree symbiotic partner is also performing more negatively. Okay, so moving on to this second question, how does connectivity predict the amount of fungal community change? I was interested in this question because so I showed the principle component analysis graphs and I think a lot of microbial ecologists sort of do the standard community composition analyses. And I really wanted to kind of figure out what the next step would be and to figure out whether we could predict how much communities change. And one framework we can think about in terms of predicting community change is this disturbance framework where if you have some some level of community composition on the y-axis, some measure of community composition, and you have time on the x-axis for instance, and some disturbance event like warming occurs at some point in time, and you measure the composition of the community before the disturbance at y1 and then you measure it later on at y2, that difference in community composition reflects how resistant the community is to changing pre versus post that disturbance. So I was interested in using this framework in combination with a relatively new method that was developed called cohesion, which is a way to quantify connectivity of taxa within microbial communities. And I don't have a ton of time to go into this method in detail, but basically it's based on pairwise correlations, so similar to network-based approaches, but instead of creating a network, what you get on a per sample basis is a measure of how prevalent and abundant highly connected taxa are within your community. And again, I said a per sample level, so you can start to apply it to this framework of how resistant our communities to change. And that's exactly what I was interested in doing, wondering whether if you start with a community that is less connected, so has less abundant, highly connected taxa in it, are those communities less resistant to change than communities that start out with greater levels of connectivity? So the plots on the right show what this framework would look like, given how connectivity might relate to community resistance. So I was interested in doing this in this experiment, but we don't have a time series, although there is this implicit assumption of temporal patterns, basically because ambient treatments, ambient warming treatments reflect sort of current conditions, and the warmed treatments reflect predicted future scenarios. So there is a little bit of this before and after temporal component in the design, similar to the cannabidiotreatments as well. So Y1 versus Y2 are going to be ambient versus warm treatments or closed versus open canopy treatments. And I measured community change between these two treatments using Bray Curtis dissimilarity, so low amounts of community change versus high amounts of community change as you move upwards on the Y axis. And I was interested in relating this to how much connectivity was in the community at starting levels. So how much connectivity was in the community under ambient treatments in this case. If we find a negative relationship, this would suggest that connectivity helps promote resistance to change and that more connected communities are more similar between ambient versus warmed treatment, suggesting that they're more resistant to experiencing compositional shifts. Oppositely, if higher, if more connected communities are less similar between the two treatment types, this would suggest that connectivity actually enhances destabilization. So as one domino falls, so do lots of other dominoes within the community, so to speak. So here I was, or what the results are going to look like, community connectivity, again, under the pre or control levels on the X axis here, these are ambient temperature treatment samples. On the Y axis is the Bray Curtis dissimilarity between ambient versus warmed communities for ectomycorrhizae and saprosophic fungi. In both cases, we see a negative relationship. Although for ectomycorrhizal fungi, there's a slight interaction between closed versus open canopy, but the overall trend is also a negative relationship. So communities that start with greater levels of connectivity in ambient treatments show less dissimilarity or more compositional similarity between ambient versus warm treatment. So again, suggesting that connectivity helps promote resistance to change. So that's the response to warming treatments. When we look at the response to disturbance, so closed versus open canopies, we find the same negative relationship or negative trend. So in both cases, there's evidence that starting levels of community connectivity help promote community resistance to change so that we can actually start to predict how much communities might change given warming or disturbance levels if we know how much connectivity is in the community to start with. Now finally, I was interested in trying to relate how community shifts relate to these functional consequences, right? Because the activity of microorganisms below ground have important consequences for sorocarbacycline. So one thing, one of the first things we did was look at how enzyme profiles shift in a similar analysis type as looking at microbial community composition. So this is a principal component analysis of the suite of enzyme activities that we measured. So similar to the microbial composition. And here we see a stronger enzyme profile shift with canopy disturbance than with warming. So you can see the open convex holes kind of slightly moved to the right in this graph. But again, I was interested in how these shifts in sort of cumulative enzyme function relate to the patterns of community responses to warming and disturbance. So one framework we can use to explore this is looking at differences in community composition between the two treatment types on the x-axis versus the difference in enzyme activity profiles on the y-axis. So basically relating the brachitis of microbial communities to the brachitis of enzyme activities. If there's a positive relationship, this would suggest there's some functional contingency in the community. So as with greater community changes comes greater functional changes, suggesting that certain members in the community do separate and unique functional activities contributing to enzyme profiles. Oppositely, if there's a negative relationship with greater community change, the system tends to remain the same or there's some redundancy in the community. So it suggests that more functional redundancy or homeostasis is what we called it. So here's what these results are going to look like. There's a difference in community composition that brachitis relationship on the x-axis and the difference in enzyme profile, similar brachitis measurements on the y-axis for just looking at how communities and enzyme profiles respond to warming treatments first. Here we see a slightly more complicated pattern for ectomycorrhizal fungi on the left. Under closed canopies in the solid line and closed points, there's this positive relationship. So this is consistent with functional contingency, basically that as ectomycorrhizal fungal communities change, so too does overall enzyme activity characteristics. However, under open canopies, there's a negative relationship. So suggesting more of this functional redundancy or homeostasis with ectomycorrhizal community changes. With saprotrophic fungi, we see under both canopy types this negative relationship, so more indicative of that functional redundancy or a tendency of the system to remain the same as saprotrophic communities change. When we look at the response to disturbance, again we see a positive relationship with ectomycorrhizal fungi, so more community change leads to more different enzyme activity profiles and no relationship with saprotrophic fungi. So basically the take home of this slide is that there is some evidence both of functional contingency and functional redundancy in the system as fungal communities change and they might even balance each other out in the end. So recapping these three broad questions, I looked at how different soil fungal groups responded to warming and disturbance to find both evidence of community shifts with ectomycorrhizal and saprotrophic fungi and importantly there's this sort of balance between mutual SND composer ratios that starts to decline with warming which could have important implications in the system. I looked at how interconnectivity of microbial communities could predict community change, finding that more connected communities were more resistant to compositional shifts in response to warming and disturbance. And finally I looked at how structure and function were related, finding some more complicated patterns where both functional contingency and functional redundancy seem to come out of the system as these fungal communities change. So the take on here is that fungal community shifts really reflected this shifting balance of fungal mutualists like ectomycorrhizae to the more free-living fungal decomposers of saprotrophs and that these community shifts can actually be predicted by the interconnectivity of taxa in the samples and that community shifts are also linked to changes in soil enzymatic function which of course have consequences for the degradation of organic matter and soil carbon cycling in ecosystem function here. So this is where I would like to end and I'm happy to take any of your questions at the end during the Q&A and I'm ready to relinquish control of my screen Sarah. Okay thank you Michael very much and again you can send in your questions and we'll get to them then at the end of the session. So it's my pleasure to then move us to New Zealand from North America and our next speaker is Amanda Black from the Bioprotection Research Center at Lincoln University in Lincoln, New Zealand and we'll be discussing old growth forests and da kauri with exotic pine plantation forests in New Zealand. Amanda welcome. Thank you good morning from New Zealand. Thank you Max and Ephemes for providing this opportunity albeit early one to present one of our studies as part of our broader program and I'm sorry I'll just see my screen so I know what I'm actually talking about and trying to look at basically the landscape ecology and some of our indigenous forest ecosystems. So this study which is the paper which this is based on is actually one of my PhD students in our lab group and apologies for that and as I described it's like a much much broader program and this particular one was looking at this all microbial diversity in these ancient forest systems with the adjacent land use changes. Her name's Alexa so I guess that you know in New Zealand we have kauri dieback which I'll introduce much later and it's one of these significant issues that we have where we have 100 of the trees that are infected have dieback and there's no cure right now and you know this is part of this whole global issue of forest disease and dieback occurring at sort of unprecedented global scales and this is exacerbated through clearance and we have fragmentation and of course biological invasions with the nursery trade and just global movement and of course climate change makes things a little bit worse and this is this is quite I think scary for us as I guess on life on earth because forests are essential for our survival and they hold much of the biodiversity of course carbon storage and this helps regulate climate change and here's just some of the examples not only in New Zealand do we have kauri dieback but across across the ditch in Australia we have jarra dieback in hawaii we have rapid or here death and ash dieback all sort of have the underlying issue of and introduced pathogen causing this. The tree species I'm going to talk about is one that's I guess pretty much around the Pacific it's actually only around the Pacific so it's a longstone ancient long-lived family of conifers it's agathas and the endemic species in New Zealand is agathas astralis there are 21 species around the South Pacific and actually quite a number of them are under attack by a phytophthora pathogen which is the orgasm I'm going to talk about this morning or this afternoon wherever you may be so the kauri forest that I'm talking about is one of our key swim forests in the top of New Zealand and it naturally occurs at 37 latitude south it's at the moment I used to cover about a million hectares and it's reduced to less than 0.4 percent of the original forest it's found now in highly modified small remnant stands and obviously a lot of them are regenerating but only there's only less than one percent of the original growth remains of these ancient systems and when I say ancient systems these trees are very very long lived they lived up to 2000 years old and so they are I guess we what we consider keystone forest ecosystem drivers and much of the associated plants and the invertebrates and animals around them kind of co-evolved and very much dependent on these trees existing and they also have a unique soil ecosystem as well these are just some of the plant species that are associated with kauri there's about 21 species completely dependent on the existence of these trees these trees are now because they're introduced by top of the pathogen they're now listed as threatened and so if these go and we have no cure then much of the ecosystem in the northern part of New Zealand will change drastically as many of these co-evolved species will become extinct as well so as I mentioned before this is part of a much much bigger program and when this when we identified what was actually happening there was many many questions that were unanswered and while a lot of the focus tends to be on the host pathogen interaction nobody actually understood what was happening in the landscape in terms of the ecology and how these invasive pathogens were changing I guess I saw microbial ecosystems including that was the land use changes how that may actually vector we call rural vectoring from one fragment to another and these land use changes in between with where we have you know commercial forestry now set up in pasture for animal production and how these actually may I guess increase the virulence of and and the spread of these diseases and so some of the questions that we're looking at is how have the success of ecological disturbances impacted on the soil microbial community and these ancient forests and their functional responses I mean I guess I should talk about what's happened in the New Zealand ecosystem and that it's compared to the rest of the world it's very very late settled in terms of human settlements and so I guess in the early 1800s we have New Zealand was it's an ark and it has birds like I had many many birds we don't have a lot of land mammals and so these birds predominantly provide a lot of the nutrient input into our soils and they disappeared as people came in the 1800s to early last century we had significant land clearance for logging but also to provide that sort of commercial pasture and prime forestry which underpins a lot of our economy here and then with the nursery trade and establishment of forestry from importing seed and plant material from around the world we had an explosion of introduced plant pathogens and the one that's afflicting Kari today is a fight offer named fight off the egg of the district in 2015 unknown its origin we suspect somewhere from around the pacific but the devastation it's causing is something of concern and so once we have these as part of this how do we like how how is all this impacting on I guess the function and the community structure of the soil microorganisms including its ability to store carbon as well so just a brief history of the emergence of Kari in New Zealand it's a it's a novel pathogen and it's associated with the tree death that was identified as fight off the egg of the district in 2015 it was actually misidentified 10 years beforehand and what is it's a soil pathogen so it infects Kari via the root systems and it has six life cycle stages which means it's incredibly difficult to actually manage and at this time we do have in boardwalks and wash stations in place to try and manage that with disinfectant it's a virulent invasive pathogen there is no life stage of a tree that seems to be immune to it whether the seedlings get infected the rickus which we call the young trees which are around 150 years old and the iconic long-lived trees are about 2000 years they also come to the disease eventually some of the vector controls we have around our forest is these boardwalks and wash stations which have a a triaging disinfectant in it and when we go around the communities and we discuss you know the possible vectoring and where you can help to prevent the spread of this we typically show these diagrams here it's like on your vehicles your shoes they can transmit these you know the spores usually the survival spores once they settle in the ground and the conditions are right they tend to germinate it's brandy release spores which go on and infect the host and we have found that this particular pathogen is not fussy but however it has a devastating impact on the kodi forest in the trees the study site of interest that we have focused on is Waipua Waipua is our largest I guess remnant growth ancient forest system that we have and it's home to some of our iconic big trees that are around about 2000 years old and these are big big trees they they store up to 30 cubic tons of carbon they typically stand they have a girth of 15 meters and they stand more than 30 meters high so they are significant trees and they exist now within these fragmented forests land use changes I'll do is I'll just change my pointer so a laser pointer here and these forest systems here are tiny this is a regenerating forest system here and this is a typical I guess New Zealand landscape in this area and it's very much pasture and we have commercial forest site here so all these little patches of our remnant old growth forest up to 2000 years old here are surrounded by all these land use changes and what the study part of the study was was to try and understand how these land use changes here with something so it's impacting on the virulence and the spread of this of this pathogen and I should say that this study was done and it's one of I guess it's a new kind of study that was done in collaboration with the traditional landowners so in New Zealand we have a lot of forestry owned by the indigenous community and traditional landowners and this was done in partnership with them the initial study which I guess pre preempted the work in this paper was looking at the land use effects and it was a very simple study published in forest biology where we took soils from around the pasture sites, the commercial forestry and the old growth remnant stands and what we did is we grew up the phytophthora agathodistata in myroclasts and we exposed them to the different soil uses that we got around the country around from the area here and what we saw is that we got a difference in a mature sporangia count and also in mature usbore count and what that said to us there's certainly something happening and they I guess either chemically, physically nutrient or biologically that is encouraging the growth of mature sporangia and usbores part of the life cycle stage of phytophthora and that certainly increases its ability to grow in these different land use changes and it hinted that there was a potential for many of these land use different land uses around these old growth remnant forests to provide perhaps a disease reservoir and for that we weren't sure so we did further sampling analysis and we looked at more at the microbial community and composition around these land use chases and we compared the two tree dominated land uses which is our old growth codifier assistance and our commercial pine plantation and our commercial pine is Pinus radiata and it has a rotational life of 25 years and so what we did is we increased the sampling sites and we only took from the organic layer a sample of 10 centimeters depth so we have one of the field technicians out here sampling we typically take a grid and the thing about codifier forests is it's very very dominated leaf litter and under there it's a much much mineral horizon this photo here is a typical it's a rare soil type it's a podzole and these form under these big trees and even if you do change the land use what you tend to get underneath here is the podzole that have formed and that's because these trees form massive massive leaf litter layers some of them have up to two meters in depth as literally their leaves just fall down and they create this leaching effect with tannins that go through and they do change the nutrient status around them and making it very hard for certain other kinds of plants to live there but it also creates this very unusual soil microbial community and we analyzed at these different sites where the soil chemistry was assessed so our usual biological valuable in nitrogen organic matter carbon phosphorus ph and we also extracted dna using usual dna easy kits alumina sequencing of the gene region and bioformatics using chym we compared the differences in alpha and diversity and composition and we also correlated these differences back to soil chemical properties to see if we could get a relationship between the difference of community structure and composition and chemical properties so i've lost my screen sorry so these ordination plots we have the fungal humanities here on i guess my left hand side and the bacterial communities here on the right hand side and the green dots represent the soil under the prime forest and the red triangles represent the soil under the kote forest and what we found is that there was a very distinct difference from fungal communities under these three species um the diversity the soil under the prime plantation had high specie diversities than the native kote soils with the kote soils having a sort of a few fungal taxa work but with higher abundance there was a significant difference in the composition of fungal communities between kote and point and that's quite obvious in these ordination plots here there was also we found the same in the bacteria species but what we also found what we didn't find in the fungal communities is that the heterogeneity of the sites also contributed to this difference so making the bacterial communities difference is not so pronounced under land use and and a little harder to interpret the results there what we did find in the um i guess the soil for the chemical properties is that we do have a difference in the carbon and nitrogen ratios and the mantel tests revealed that that was one of the explanatory variables with that and organic matter correlated to the differences in the fungal communities but for the bacterial communities there's more around about um biologically available nitrogen and total carbon in the composition so we on the um on this side here we have the relative frequency of the fungal taxa that we saw and it's a little bit small but um basically on this side we have the soil taken under the kote forest and on this side we have the soil taken under the pine forest and there were a total of 18 fungal filer in um these soils with probably about five percent that were unassigned uh escomicota and basidiomicota formed the majority of the reeds escomicota was certainly higher in the pine here where bascomicota was certainly higher in the cody soils and if we go over to the heat differential heat tree reeds that we see in the blue nodes here represented the high counts in the pine forest and in these um orders sorry half my screen is covered by the um the menu for the for the webinar itself and the green and the green nodes here were certainly the um dominated by um the cody forest and so what we did see is that um the major fungal taxonomic groups were significantly differences in their relative abundance again so the green ones up here were the cody forest and their hair were the blue ones and what this is it's kind of a reconnaissance something but what it's uh I guess further demonstrating in these diagrams is that they were quite distinct and significant differences between the fungal communities and the land uses in pine soil versus um the cody soils again in the bacterial communities to be looking at the um the frequency relative frequency diagram here we have cody on the side on this side here and we have pine on this side and in the bacteria we had 51 species uh phyla recovered uh I guess the majority of these were significantly low so well we had quite a lot of phyla we didn't have I guess a lot of abundance and the only the 20 most abundance we actually show in here and again the same for the um the um sorry the tree we have green shining here the nodes for the cody soils and we have the blue here and I have to guess because my screen's half covered um and over here as well as for the pine soils and while we do see these differences in the bacteria and between the land uses that again it's this relationship is not so strong as we see in the fungal community changes so I guess the story is a little more complicated and and more difficult to interpret and it would require further um exploration about what this actually means in terms of land use changes but also with the uh I guess the introduction of these pathogens which would they get the visitor and I just wanted to introduce this uh it's another paper but it's also part of this biggest study it's around the community metagenomics that we looked at in these particular sites and so we went from comparing wine as the land use and and cody as another land use but we also looked at the the differences between in these communities where we had uh I guess infected trees versus um non-infected trees and what we did find is that that there was a significant difference in the biochemical uh biogeochemical cycling genes particularly in the carbon degradation gene so we find that um and and infected sites we had um complete differences in the carbon degradation genes then we did it the uninfected sites and the implications here are disturbing so I should have said that the the read here represents the asymptomatic or uninfected sites and the blue is the symptomatic um which is the trees that have obvious disease symptoms shown and it has implications for carbon storage and certainly carbon storage within the soil system as well and this is something that uh we need to it's something we haven't actually considered in in the system is like how do these pathogens not only land use changes but how do these pathogens uh influence uh carbon cycling in these systems but some of the take home um I guess discussion points here is that the altered soil micro communities that the forest type exerts strong influence on the diversity and composition of the soil communities um sorry I'm just gonna I can't actually read my screen it's terrible exerts a strong influence on the diversity and composition of the soil and the soil fungal communities kairi were certainly more well defined in the community and dominated by a few key groups and the differences in the soil chemical properties did uh certainly align with the differences in the microbial communities and helped explain some of the differences that we saw in the forest types and how does this link back to die back well also interestingly we didn't find any of the pathogen in the the pine forest that we sampled and what we did find when we did have a there's a table on the paper that looks at the the different I guess the trophic levels of between these two systems and we did find a lot more saprophytes that existed in the kauri forest a lot more uh symbiotrophic fungi happening in the in the pine forest and the implication here is that with all these land use changes in the introduction of different plant species that there is a potential that we have lost a lot of our protective fungi to I guess protect against these invasive pathogens and and how does that I guess relate to inform particular management plan when it comes to you know is there a certain remnant size of these land use changes that we can certainly identify as they any smaller than that and they're under I guess more threat to these invasive pathogens with the loss of their I guess their protective fungi and and how does this in these changes interact and we look at restoration of these old when we look at regeneration restoration of these kauri forest how does that influence their ability to I guess resist these pathogens as well so the future is here is part of the program comes under I guess the interest here was we have a billion tree program I think that happens a lot around the world where the we look to plant more and more trees to offset a lot of our carbon things but if we are losing a lot of our protective fungi in the soils then then that has implications there perhaps that we need to look at the type of tree species that we plant we also need to look further and see what actual properties and and microbiota are positively associated with soil health and we also need to identify the other environmental factors their ability they have the ability to defend against this pathogen I'd like to acknowledge that this was funded by a tertiary education commission and in collaboration with Tororo who are the indigenous owners and community that we work with clarity um in collaboration with thank you and that was me um I apologize I couldn't see half my screen but hopefully that you've got I guess the key messages there and of course there's the paper that you can look at over to you max Sarah thank you very much Amanda and uh we'll come back to I think a number of question points that are also coming up already and I also have things to ask further about very interesting so we will quickly jump to Europe and our next speaker is Fleming Eklund from the department of biology at the university of Copenhagen and he will examine a different kind of a disturbance this is again the response of wood ash application in both agricultural and forest soils so Fleming thank you yeah thank you I'll do this presentation of of this paper that was previously published in in famous microbiology ecology which is called total on our sequel in a sequencing reveals multi level microbial community changes and functional responses to wood ash application in agricultural and forest soil and I'd say that it it's uh it's part of of of of a much larger project that we did in our center for bioenergy recycling ash bag um and then uh I think if if you see the title you may want to ask two questions and the first question would be why should we investigate wood ash and that is in in at least in in our part of the world it is a very relevant thing because a lot of our energy it it comes from from wood this is disputed at the moment many people think that it's actually not a very green energy but still this is a fact that much of our energy comes from from from from wood and that there are several issues associated with this wood ash one thing is that deposition of wood ash merely as a waste is it's costly and it's also a problem that that there are lots of essential plant nutrients in wood ash which are just lost if the ash is just deposited so it may be a good idea to recycle the wood ash to to to reuse the the plant nutrients it's also an issue however that that the wood ash may contain heavy metals and that it has a drastic effect on soil pH but what we did in this uh in this ash bag center was that we uh investigated possible negative side effects of wood ash recycling in various ways this is one of the of the papers that came out of it and then question number two would be why should we apply this total RNA sequencing instead of more conventional methods it is because it will allow to simultaneously assess all parts more or less at least of the soil microbial community prokaryotes fungi micro eukaryotes that I have worked a lot with myself and which are often overlooked in in in microbial ecology also it it will allow to at the same time to detect the expression of functional genes if we if we more or less get the full RNA picture out of the soil and it has the advantage also as compared to more conventional DNA based methods that we have no private bias as we have in the DNA based uh methods and this is what we did as it also says in the title we uh we we uh looked at two different soils uh a loamy sandy agricultural soil and a humic forest soil the o-horizon and from these two soils we made microcosms in triplicates from 50 grams of soil and we mix this soil with wood ash to different concentrations that is zero three twelve and 90 tons ash per hectare corresponding to these to these amendments and it is essential to say here that the normal amendment to to to a normal soil if you applied in a forest would be three tons so so we used also some quite unrealistic uh doses which I think is necessary if you want to do an experiment like this because otherwise you may not see the effects that are there when you apply small doses but may not be very clear when you only apply the small doses then in these uh systems we destructively sampled on day three ten thirty and hundreds and we measured then uh four different types of parameters we measured physical chemical soil parameters uh I'd say pH should be mentioned first pH electrical conductivity uh we measured organic carbon and we measured nitrate ammonium and phosphate then we did a more conventional quantitative PCR to estimate the abundance of bacteria and fungi and then we measured these pools of RNA ribosomal RNA that is to to to investigate diversity and richness and we measured uh messenger RNA to get some idea about uh different functional genes expressed in the systems that we work with and these are I'd say some of the results that I present here and the first one is the physical chemical soil parameters we saw not very surprising we saw a large increase in pH in the soils and it is uh because it it uh it influences the results that I'll show larger you should notice that this the the y the scales on the y-axis is here are not the same the upper graphs here show the pH that that uh that we reached a much higher final pH in the agricultural soil that we did in the forest soil also we had an increase in conductivity because of all the different ions that that we uh that we amended the soil with also uh we got an uh increase in dissolved dissolved carbon also which is probably primarily because of of of simple physical chemical interactions uh with the soil that the ash facilitated we then we got an increase in ammonium which is which is probably due to various various microbial interactions this ammonium increase is not as clear as the increase in some of the other parameters because the microbial interactions are complex and similarly we can see uh we can see that there is an effect on on nitrate which is even more more mixed than the effect that we see on ammonium and then also we see an effect on on on on phosphate which is also not straightforward probably because that the the the effects on on pH will affect the solubility of the phosphate we uh we I haven't shown that but we we analyzed the results and this analysis showed that that the that pH conductivity dissolved carbon and phosphate were the main drivers of the microbial changes that we saw in the systems and that I'll show now the first one is the QPCR and we can we can see here a picture which is which is similar in all the microbial uh all the microbial measurements that we made that uh that we have I'd say uh more or less straight not completely straight straightforward but more less straightforward uh effect on the micro positive I'd say effect on microorganisms in in the forest soil because there we reached after the ash application we reached some pH levels that are favorable for the for the microorganisms that that we that we looked at and also here you should notice that that this the scales are quite different on on on the graphs but on the other graphs here we we have bacteria as measured by 16 uh there's INA genes and below we have fungi as measured by ICS and you you can see that in the forest soil where we reached as I would say the favorable favorable pH values we have a clear effect but but no really clear effect in the agricultural soil except that we see that the bacteria they they were actually diminished by this treatment it should be noticed though here that this is QPCR and we are we do have here this private bias then we we look here at the results from directly measured by the ribosomal INA where we have no primal biases and what what we see here are relative frequencies of the different types of microorganisms we firstly you may notice that in the first in the upper panel here we have all groups of organisms and this is 0, 3, 12 and 90 tons ash per hectare and you can see that there's no 90 tons ash per hectare in the agricultural soil because something happened which prevented us from from look at the INA as as you saw on the previous graphs it's not because all the organisms actually were eliminated by the high pH but we were not able to retrieve any INA overall and probably not very surprisingly if if you look at this panel which shows the bacteria here we could see that overall amendment with bio ash stimulated the stimulated the stimulated the copiotrophic bacteria whereas there was the opposite tendency for for the oligotrophic bacteria that they declined at least with high ash amendments and you can also see that in maybe a bit surprisingly that in the 90 tons per hectare treatment in the forest soil we saw an increase in number of fungi but it is worth mentioning here that it's it's only a particular group of fungi that actually carries most of this increase and then I have here on the next slide I have taken from the previous slide the the two lower ones the two lower panels from the previous slides to to show in in in more detail that we saw for all systems just we saw an increase in in the number of micro eukaryotes meaning mostly protozoa with time here in the agricultural soil and the three ash amendments and here in in the forest soil and this general increase I would I would tend to think that this has not so much to do with the experimental purpose but rather that when we we have this I would call the closed system effect that we have a certain micro succession when we make a closed system a closed system and here we it's not unusual to see a micro succession here ending up with a lot of graces in in the end but in in in the forest soil that there was also this effect was also overlaid by what we were looking for that is that we could see that this micro succession was stronger with higher ash amendments then the two lower panels here show total for all groups over all groups of organisms that is fungi micro micrograces bacteria etc what we looked at show how this how the treatments and the time affected richness and and the autographs and diversity you can see that pretty similar these not completely but pretty similar these patterns we see here and and you can see at a general decline with time in the systems where in in the agricultural systems which is also I think a result of this I would call closed system effect that that things happen in a closed system that are different from from a normal system but but but we can see that in the forest soil a similar thing we did not see quite the same pattern here which is probably because that the organisms that we focused on they were actually stimulated by the ph change that the ash promoted then to the to the functional teams we did find as we expected an increased transcription of presumed stress response teams at but only at the very highest level of of ash and that is that we we find we found teams associated with sporulation which is bacterial sporulation which is a thing that takes place under unfavorable conditions also we found we found an increase in in membrane transport of proteins which we believe was a result of the changed osmotic conditions and the changed content of toxic metals in the system and finally we found an increase in chaperones that are genes that ensure correct folding of proteins and are involved in in in the in house cells they cope with the stress induced denaturation of proteins and then and finally a few conclusions we did find as we hoped to find that the social RNA sequencing allowed us to at the same time follow bacterial fungi and bacterial feeding protozoa in using the same method and in especially this pattern was not clear in the agricultural soil but in particular in the forest soil the high astrosis which caused I would say appears to increase to a moderates but higher level we found an increased bacterial growth which the protozoa responded to and this final we finally resulted in a decreasing fraction the relative fraction of bacteria in this system and also we found that analysis of messenger RNA demonstrated increased transcription of presumed stress response genes at 90 tons ash per hectare I should I should have added another line which is that this this method actually provide us with some quite astonishing possibilities which we are more or less not able to use at the moment because really many of the results that we get both in terms of ribosomal and messenger RNA we're not able to interpret them because we don't know actually what the what genes and we don't know what organisms that we actually find represented in the samples this will hopefully improve in the future and then finally I will thank the Danish Council for Strategic Research for supporting supporting our sender and I will thank the persons already named on the first slide but also the many other good colleagues that from from ash back and I would in particular like to thank my former colleague and the head of our ash back center yeah thank you that was that oh thank you very much Fleming and indeed thank you to all our speakers Michael Amanda and Fleming and so we will open this up for question and discussion and before that I also want to thank both Sarah McKenna and Joseph Sherylworth who in the background are making sure that everything is working well and all the connections are in place so thank you but again Michael and Amanda if you'll join us as well then we'll look into the questions so this is one actually for Michael do you have plans to test if the enzymatic profiles map to the genomes and the reason this is Joe Sariva who's asking is because it has been suggested a positive correlation of functional redundancy contingency with microbial diversity yeah that's a great question at this point we don't because our I came into the project when the data had already been kind of assembled and the enzyme assay was already completed and the amplicon sequencing that were already run so I think that there is some transcriptomic and sort of more genomic based functional data that's being that is starting to come out of the before warmed project but as far as it relates to my specific role in the project I don't have any any specific contribution to that to that part so we're left with the enzyme assay and but the the person asking the question is completely right that it's it can be very difficult to link the sort of compositional profile of a microbial community from amplicon sequencing to the functional potential with the with an enzyme assay which of course measures total potential activity and not necessarily maybe the most critical or or sort of real aspect of the functional potential of the system yeah that's a good question now it is actually a follow-up question and also a similar one from tamar bar k relating to biomass of fungi to bacteria and also I mean could the species interactions between fungal groups and their surrounding bacterial partners be important um yeah that's very possible so I imagine that the fungal to bacterial biomass ratio probably shifts we don't I don't have access to that data so that's not something I can answer specifically but I think we're calling back to other sorts of forest warming experiment papers that I've read I do remember people have noted shifts in fungal to bacteria biomass ratios so I guess I would imagine sort of bacterial biomass to increase relative to fungal biomass in this system because past papers have shown declines in in fungal taxa like specifically ectomycorrhizal taxa I forget the second part of the of the question but oh yeah well this is the then the interactions between bacterial fungal communities as well yeah so I have to study them I looked at that briefly when I was I did some sort of kind of basic network analyses to see if there were um interesting like inter can inter kingdom uh correlational patterns between bacteria and fungi and there did seem to be some different correlational patterns between the warming treatments like different groups of fungi were differently connected to different groups of bacteria but it never couldn't really get a clear sense of what that would mean but certainly it seems possible that these microbes are interacting in in ways that I wasn't able to test with the data that I had but certainly could be important for determining their compositional shifts or functional responses yeah I have a follow-up question I mean both to you Michael but also to Amanda on thinking of one we're looking at the soil microbial community structures bacteria fungi and so on but how much of that whether it's the temperature effects or of course then when you have died back of a key species is really the changing vegetation that might be driving what's happening in in the soil community and how do you start linking to that and did you follow that for example Michael on I did not there's a paper by chris renanda as in global change ecology that did link um changes in ectomycorrhizal uh community composition and taxonomic responses to changes in tree species performance using photosynthetic rates so there is some past work in my experiment linking microbial responses to physiological changes so I was kind of building off off of that work um maybe Amanda you could talk about how that applies in your in your system it's uh you know it's a really good question trying to understand what is actually driving what um you know when we get pathogen invasions obviously um these trees become infected you're going to get a change in in carbon signatures and inputs into the soil but also you're going to get um a change in leaf litter composition because you know when sick trees uh they tend to drop different things and so that potentially could be driving the community shifts that we see or it could be the invasion the pathogen driving the community shifts that we see we haven't been able I mean that's that that will be something we'd like to do but that's you know a study that I think needs to be done but the problem with doing it in the field is like we can't set up field trials with the organism that we illegally can't we have to do it in the lab and then the trouble we're doing in the lab is that um because these trees are so long-lived the systems that we replicate and mesocosm stuff just don't give us that clear picture and we've tried it with seedlings and we just it's just all muddled and murky but a really good question and that's something that we'd like to look at in future. Okay let me go to ash additions and so there's two question related to that uh one of the cumulative effects on continuous additions of ash to these soils and then also the impact of cadmium of naval bacterial communities so how is this affecting in terms of this is a one-time addition or is it's going to be repeated what what what what what people uh normally suggest is is that you do it on on a regular basis but regular here I think it means as far as I remember it's uh it's it's three times in 75 years or something but the thing is that that that these pH effects and this is very this is very complicated actually because at least if you as as I showed it may have some effects and I think I wouldn't normally recommend people to to do it in an agricultural soil it has been attempted but but normally you would do it in a forest soil and I don't think that is I don't I don't think there's any doubt that it will normally stimulate production in the forest but the thing is of course that the forests are very different but by if you have a forest that is worthy of protection or something it may be a very bad thing to to to increase the pH the and and and as I said the pH effect will last a very long time so if you if you repeat the ash amendment then you may in in the longer term increase pH that is that is one one thing yeah I would like to add I think to this that that when I I said 75 years normally when we talk about forests the time perspective is really really long and and my guess would be that in 75 years we do have quite other ways of providing society with energy than using so it it is a bit it's a bit a strange discussion this one I think uh the the the other question was about the cadmium and how would would would that affect how would that affect things you could say that if you do this in in a proper way which we often do not do but if you did it in a proper way you would actually only recycle ash in areas where you had harvested wood then there would be no problem because because you would only bring back what you actually took from the forest in the first place so so so there would be no problem but the thing is that at least here in Denmark we are not doing it in a proper way because we import we import biofuel which is in my opinion that's a political question not a very good practice and in that case you may increase the cadmium amount in the soil still we have investigated that in detail in in in other sub projects and actually it there seemed to be only a very slight problem because it's it's not bio available the cadmium that apparently that you return okay yeah that was far too long okay well there's a series of other questions more broader relating to really how to measure and analyze microbial diversity and functions in soils and this is really for everyone and it's actually a could be a topic of its whole own for for a webinar but the approaches I mean how what we should do there's one is how much is it worth to invest in apricon sequencing giving the amount of information retrieved is limited to ASVs should one focus on metagenomics or other omics even if the cost is higher so yeah maybe a discussion with everyone how does it fit to your specific questions but then also broadly how do you do enzyme assays how do you link that with who is going up or down in abundance so maybe michael you want to start yeah yeah i'll kick things off um in my case i've worked a lot with sort of traditional composition-based microbiome datasets so asv's or otu's or amcon sequencing type stuff i've been thinking about this type of question for a while i think what the sort of position i've settled on is that it is best to try and have some sort of a priori idea about what sort of function you expect to change in the system or or how it might change across the gradient that you're studying so it so in our case in this in this forest ecotone system we expected fungi to be important because they play an important role um symbiotically with plants but also in terms of accessing hard to access carbon and nutrients from the lignified plant organic material so we expected that enzyme profile shifts to reflect sort of a change in peroxidase or polyphenol oxidase or other sort of hydrolytic and oxidative enzymes and we do see that i wasn't able to get into it into the paper but we do we do see it more of a direct link between fungal changes and the types of enzymes that are important for their their degradative capabilities so i my personal approach is to try and make those links ahead of time and expect to see where uh functional changes might be occurring but of course as i'm sure minda and flammey are gonna say there's drawbacks to any approach right the enzyme profile gives you a cumulative sense of maybe potential activity but it's not linked to organisms and metagenomics and transcript dominoes and all that kind of stuff or you get more of the genetic aspects but it's harder to tease apart how that's actually functioning in the soil environment i mean i can pass that to amanda and you can talk about that well can i just be successful to say that you should actually mean start with a hypothesis yeah and then go in and look at what methods would be appropriate yeah and that in certain places that's easier said than done when if you need to go in and investigate what patterns are there just to just to get a starting point but yeah that that is my traditional approach uh yeah no same in the case of this this study that we have this wider program which is five years um we had to do a bit of that i guess reconnaissance supplement to try and get a handle of what patterns there may be out there and narrow that down i mean we all know with our scientific background and i guess our disciplines that we can guess which which functions we'll be looking at in the case of you know forest i bag you know the the carbon's going to be affected and so you can start to narrow down on things like that you can look at in general community system we find PLFA is quite good so you know your general your general stuff and then and then refine your questions from them you suspect that the carbon degradation is going to be affected and we did just a geochip so semi quantitative analysis of the geochip and then from there you can narrow down and try and tease it all together but i guess it also depends on how big your budget is that someone seems to define that a lot more yeah plumbing you anything to add on on the approaches yes um two things actually i'd like to add but be firstly i think your question is um i think your question is difficult to answer because these methods are actually developing so fast that while we talk about it here it may show up that that another method has been developed which is better there's a tremendous uh there's a tremendous development here and also these methods become cheaper and cheaper so so so that that's one thing so it's it's difficult to give which is i think a frustrating and also interesting but but but but but but but also i would say that but but but but i'm actually much of an organism person uh i think that that it's a pity that it's it's it's difficult i think to get funded to to to make more basic research to do this interpretation how what do these organisms actually do it's much easier to get funded to make a broader a broader investigation which sometimes i would say does not lead us much further than basic basic work in many cases be much better no i mean there's definitely so many organisms that we don't know yet what they do what their functions are so even if you have an asv or a 16 star in a full sequence that still doesn't tell us then what are they up to no yeah yeah we're only as good as the databases that you can scaffold contents on to and all that kind of stuff so that's really important to consider as well okay i think we need to actually wrap up for today or for others it's just the beginning of the day or for for amanda but so thank you for getting up very early to join us and uh flaming and for staying up later and this has been really good we've had a good audience from around the world i was looking at this as well in terms who's tuned in so thank you very much this was a wonderful discussion really good presentations and we'll see each other again with the next webinar probably in early january so for that take care everyone and and again thanks for for joining and again if you missed earlier webinars they are available on both the oup and fems websites as well and more to read about soil ecology as well so thank you very much and take care