 Good morning, good afternoon, good evening, and I know for some a very late evening. I'm Max Hegbel-Mitterey in chief of FEMS Microbiology Ecology, and it's my pleasure to welcome you to this webinar on microbial ecotoxicology. FEMS, the Federation of European Microbiological Societies, invests in science, using the income from our journals to fund charitable activities and support our community. So we provide grants to scientists, organize and support conferences, and sponsor a range of events such as this webinar series, which provide a forum for the presentation and discussion of key research enabling the flow of ideas to continue despite the current cancellation of in-person events and conferences. Each month we are highlighting a different topic of microbial ecology. If you missed earlier webinars, they are also available via the FEMS and OUP websites. Before we begin, I also wish to thank the staff of FEMS and Oxford University Press for all their work in making these webinars happen and the behind the scenes detail that's so important. Today we focus on the topic of microbial ecotoxicology. Our speakers will highlight some of the fascinating questions and the interdisciplinary investigations of the response of the microbial compartment and ecosystems subject to environmental contamination. We have an introduction to key questions in this emerging area of microbiology ecology research by Stefan Boulimer, followed by presentations by Waymin Sun, Demitrius Carposas, and Karen Wojc, who will explore microbial adaptation in anti-monic contaminated soils, the response of soil-philosphere microbial communities to repeated fungicide application, and then how green infrastructure and atmospheric pollution shapes urban bacterial communities. After the four talks, we will open the session for your questions and for discussion on this. So please submit your questions via the question link. So with that, it's my pleasure to introduce Stefan, Waymin, Demitrius, and Karen, and first, Stefan, I'll give the floor to you and you can take the lead on introducing us to this topic. Thank you. Thank you, Max, for the kind introduction. Can you hear me? Can you see my screen? Yes, we're good. Okay, great. So let me start by stating that microbial ecotoxicology is not a discipline like microbial ecology, but it's a research area which is very interdisciplinary. It associates microbial ecology, chemistry, toxicology, and the idea is here to understand interactions with pollutants and with each other in communities, looking at the molecular level, the organism level, the community level, and the ecosystem level. I guess the main point here is that we also have a goal which is to put microbial contributions to ecosystem services on the map for the general public, but also for policy makers. And so my job is to give you key questions, but I guess we first need to also define the key challenges. And one of the main challenges is just human activities. And human activities affect many things, but they also affect microorganisms. And I would like to point you to this review by Kavi Kirli et al. And it's about climate, but not just about climate. As you see, these human activities will have a lot to do with toxicity as well. I have a pollutants or fertilizers and so on. And so I won't set too many questions. I guess I just want to propose a very generic one, which could be how do microbial communities respond and adapt to toxic perturbations. I guess this applies to all the free talks today. It's quite generic. The key point here is these are questions of microbial response and adaptation. And of course, now we have lots of omics which make possible studies in unprecedented data. When we look at the environment, these responses will depend on pollutant availability or on the capacity of a microbial community to transform them. These responses will be affected and maybe also masked by variations in conditions, including hydro dynamic regime, toxicants which are present, their mixtures, cocktails, concentrations and so on. And also because biologically speaking, there are many different modes of response and adaptation, toxic perturbation. And if you look at the classical ones like resistance, tolerance and persistence, I want to point to this review here of 2006, which explains these things very clearly in relation to antibiotics. There is another one which I think is very important with regard to ecosystem services and this is resilience. Resilience, of course, is something which has to do with the maintenance or gaining again a function within an ecosystem. And so because of all these complexities in conditions and modes of adaptation, the question is how we can disentangle or tease apart all the different factors when we look at microbial technology. One way to tease apart different factors is to try to control environmental fluctuations, maybe during lab work with mesocosms. And here I just want to show something that we do in Strasbourg. This is a 2D lab river where we sheet in polluted water and we look at fruit space and the evolution of microbial communities as a function of bacterial degradation of hydrogenated pollutants. And this was set up by Guiney Linfeld and we have in Strasbourg, CNRS and we have a good collaboration ongoing for many years now. Of course, we can stay in the lab and do try to look at the active layers to try and make simpler communities. I think another thing that we need to do more is maybe look at the interactions from the molecules that are used for interactions between different microbes. But we also have, as I stated earlier, key urgent goals which are operational in nature. We want to put microbes on the map also in the positive sense, especially today when we are in MEICANN. We need to state again and again that microbes actually contribute positively to services. And we need to convince policymakers that microbes can help to develop policies, develop generalizable predictive models for risk assessment. And we would like to be able to include microbes to define quality standards. At the moment, this is not the case. And also we can use microbes as indicators of toxicity and maybe they can be very useful to predict more accurate no effect concentrations. And if you are interested in these questions, I guess the paper by Stephen Bess will be of interest to you. So we need to make microbes visible, but we also want to make the scientists doing the science visible. And here I have to put in a plug for a network which exists. And there have been already two conferences. This is the first one in Lyon in 2017. And there was one where we have no picture in 2020 because it was online. This is a community of scientists. And it's quite international even if it was started in France, more than 118 members by now from 35 countries. And so you can go to the website and register, and it's completely free. And then you'll get all the information also for the next microglobal ecotoxicology conference, which will be in 2022 in Montpellier. And so I guess this also helps to put microglobal ecotoxicology on the map. As you can see, publications which use exactly this term are increasing, and they are also increasingly cited. So getting back to today's key questions, I guess there are several questions which are maybe more precise than the more generic ones that I mentioned before. Waimin is going to talk about antimony. And I guess his one question, what one could ask from his paper, which like the other two speakers, the papers were published in the two last years in microglobal ecotoxicology. So how does a minor contamination effect so in microglobal communities? And metabolic potential. This is really interesting. It's not just about tsunami as a function of soil depth. And so there are issues of nutrient content and biomass and the type of, and the extent of contamination as a function of vertical profiles. And so the second speaker of today, Dimitrios, was actually a speaker also at the ecotoxicomic Converses. And he is interested in pesticides or fungicides in particular. And in this case, I prodi on, and he will talk about comparisons between soil and the field sphere and about negative and sometimes positive effects of pesticides may differ in soil, which is well studied and philosophy, which is well, less well known. And so here we will hear about key microglobal players and I guess the impacts of pesticides on ecosystem function. And last but no case speaker of today will be, and she will talk about the importance of certain a biotic and biotic parameters on the microbial communities in an urban environment. And here this interplay is clearly shaping microbial diversity and sometimes in unexpected ways. And I guess this will be also quite interesting to everyone today. So I guess we're in for a couple of or a couple of free, very interesting and diverse talks today. And with that I hand that Thank you, Stefan, a very nice introduction to the questions that we have in store for for today. So I'm pleased to introduce Waymin Sun from the Guangdong Institute of Eco-Environmental Science and Technology in Guangzhou. And again, as was already introduced, is going to talk about microbial adaptations to anti-contamination. So Waymin, welcome. Thank you, Max. And thank you, Stefan, for introducing my presentation. So I'm Waymin Sun from Institute of Eco-Environmental and Soil Sciences. So today I'm going to talk about the story regarding the microbial adaptation in antimony-contaminated soils. At first, I would like to introduce some research background about antimony. So antimony is a naturally occurring toxic metalloids. So the antimony and arsenic belonging to the group, belonging to the same group in the periodic table. So if you take a look at the periodic table, they both of them belong to the group 15, and they are neighbors in the periodic table. China accounts for more than 90% of the world's antimony production. So China is also the world's largest antimony-producing country. But antimony toxicity is strongly dependent on their reduced species. So we know that arsenic can be biotransformed by microorganisms. The microorganisms can transform arsenic by reducing by reduction, oxidation, de-methylation, and methylation. However, our knowledge regarding the antimony biotransformation is still very limited. It's not very clear how the microbial populations develop in response to long-term antimony pollution, and which geochemical factors affect the microbial composition in antimony-contaminated environments. So we have performed some previous studies, and we found some very interesting observations. For example, we found antimony-related parameters show substantial effects on the... let me put this laser point. Yeah, antimony-related parameters show substantial effects on the microbial diversity and the community. Here is a result from a random forest model. The RF models predicted that some antimony fractions, these are antimony fractions, show substantial effects on the microbial diversity. This is a structural equation models. We found that some antimony concentrations show substantial effect on the microbial diversity and also the microbial abundance. So we also observed that the metabolic potentials of some innate microbial community was influenced by the antimony fractions. So here's a key map showing the correlations between some antimony and arsenic-contaminate fractions with the... and arsenic-related genes. You can see here SB5 and SB3 showed significant and positive correlation with many of the arsenic-related genes. This is the result from another experiment, from another project. So we found... this is a metagenome from antimony timings, and this is maybe too small to see, but I can show you this is the arsenic-related genes, and we found the presence of a lot of arsenic-related genes in the antimony timings. So now here we come to the first scientific questions. How antimony contamination shaped the microbial communities? In order to answer this scientific question we perform, we investigate the effects of antimony on vertical soil profiles. We selected three vertical soil profiles from the largest antimony mining area. So that is the Shiquan Shan mining area. So we selected three soil profiles. Two of them are heavily contaminated, and one is uncontaminated. So we categorized the death-resolve soil microbial community and investigated the microbial community of the mining contamination... investigate the effect of the mining contamination of the microbial adaptation. So we can see the... from the geocampal profiles, we can see that contaminated soil profiles show the distinct death-resolve effects when compared to uncontaminated soil profiles. So this... if you can take a look at the box plot, you can see the fractions of antimony are significantly higher in the contaminated soil profiles. The microbial community are also totally different. And you can see this cluster is from the grade that's represented, the microbial community, from the uncontaminated soil profiles. The red and orange represent the community from the contaminated soil profiles. If we take a deeper look at the data, we can see that the soil death increased, the concentration of antimony gradually declined with the increase of the... sorry, I cannot see this one. Declined with the contaminated soil profiles. So you can see that the yellow dot represents the concentration of the different antimony fractions in the contaminated soil profiles. You can see the decreasing trend in the contaminated site. So we also observed that antimony contamination reduced the alpha diversity of the microbial community in each soil profiles. And if you can compare this alpha diversity index from the contaminated soil profiles and uncontaminated soil profiles, you can see the alpha diversity from the uncontaminated soil profiles are significantly higher than the guys in the contaminated soil profiles. The RF model also suggested that the low alpha diversity could be attributed to the contents of the total concentration of antimony. So we performed the RF model on the channel index, and we can see that the SBTOT is the most important factors affecting the channel index. So we then performed the microbial interaction networks at different desk profiles to see how microbial communities changed along the soil depth. And we see that microbial interaction in the UCP, so that is the pristine site, remained this is the blue networks. The blue networks remain relatively stable while it showed an arboreal exchanging patterns in the yellow one you can see. The microbial interactions were loosely connected in the heavily contaminated surface soil but gradually recovered and were well connected in the less contaminated deeper soil. So the network suggests that individual species become more connected with other partners to perform maybe some centrifugal functions in less contaminated soil. So in order to verify this hypothesis we performed the metagenomic to see the metabolic potential of the microbial communities in different soil depth because I don't have enough time to show you the other data I just show you the data of the arsenic related genes. So we found the abundance of arsenic resistant genes decreased with the soil depth. So this is what we got what we learned from the project investigating the effect of anemone contamination on the different soil depths. So here we come to our second scientific question so who can transform anemone? So this figure shows how we how we use the combination of DNA seed and the metagenomic meaning to identify arsenide oxidizing bacteria. So in this project we also use this platform to identify the bacteria responsible for antimony biotransformation. So we investigated we tried to identify the bacteria responsible for anaerobic antimony oxidation in paddy soils. So the rice tends to absorb more antimony than other cereal or other plants. So among the rice the SB3 the rice are more efficient in of uptake of SB3 the SB5. So if we can oxidize the antimonyte that is SB3 to antimonyte that is SB5. If we can oxidize SB3 to SB5 then we can reduce the absorption of antimony by rice paddies. So we check we want to investigate the occurrence of the antimony oxidation in paddy soils. So however the paddy soil is a reduced environment which means the oxygen is absent in the rice paddies especially in the flooded rice paddies. So the microorganisms may use nitrate as an alternative electron acceptor to oxidize antimonyte. So we try to investigate the occurrence of anaerobic antimonyte oxidation in paddy soils. So we set up four different treatments of microcosm to investigate the search occurrence. So the red treatment represents SB3 plus nitrate. The blue one SB3 and the orange one nitrate only and the green one is a sterile control. So oxidation of antimonyte to antimonyte was only observed in the treatment amended with both antimonyte and nitrate. If you can take a look at this figure you can see the decrease of the antimonyte, antimonyte SB3 and with the increase of antimonyte over the course of incubation. However you cannot see this trend in the blue or gray treatment. The blue one is the we provided SB3 only and this is a sterile control. We can also see a decrease of nitrate with an increase of nitrite in the red treatment. So this observation told us that anaerobic, anaerobic antimony oxidation is driven by microorganisms and the amendment or the addition of nitrate may facilitate the anaerobic antimony, antimonyte oxidation. So our next step is to identify the biomarkers responsible for this process. So given the chemical, the similar chemical structure between arsenic and antimony, so we propose that microorganisms may use similar pathway to transform antimony as arsenic. So we selected the AIO agents. This is a gene for arsenide oxidation. So we propose that the AIO agents may be responsible for antimonyte oxidation. So we also set up three different treatments and we found the copies of the AIO agents significantly increase throughout the incubation in the treatment amended with antimonyte and nitrate. However if you take a look at the blue or orange bar, this is the SB3 only or nitrate only, you can see that no such change in AIO agents abundance. The AIO agents abundance actually decrease a little bit in this case. Also we found a significant and positive correlation between the abundance of AIO agents in the treatment amended with the SB3 and the nitrate and the concentration of SB5. So in another case, in another study, we performed the RNA, RTQ, PCR, and also we observed the transcribed AIO agents showing a positive correlation with the concentration of SB5s. So all these evidence indicated that AIO agents may be responsible for the antimonyte oxidation. So now we have the, we know microorganisms driven this process and we have the biomarkers. So we can perform the DNA to identify the antimonyte oxidizing bacteria. So we performed, we set up four different treatments. So this is C13 or C12 CSBN and C13 C12 CN. So in this case, we provided C13 or C12 labeled biocarbonate plus SB5, SB3 plus nitrate. In the control treatment, we only provide a C13 or C12 biocarbonate plus nitrate. So if you take a look at this figure, so we can see compared to the C12, C12 SBN. So this is the black, the black lines. The highest abundance of the AIO agents gradually shift to the heavier fraction. If you can take a look here, it gradually shift to the heavy fraction. It is more obvious in the later date, in the later time point. So this implying that nitrate dependent SBO be incorporated, the C13 carbons over the course of the incubation. However, if you take a look at the controls, we didn't provide antimonyte. So there's no shift during the whole course of the incubations. So then we selected, I labeled here, we selected the representative fractions for ampiconsequency. We characterized the microbial community of the representative fractions, and I can show you the microbial community compositions in each fractions. So the bacterial enriched in this case in the C13 SBN are of great interest because this bacteria might be responsible for the SB3 oxidation, antimonyte oxidation. So finally, I highlighted here, we found bacteria that bacteria affiliated with Azoacus dominated in the heavy DNA fractions of C13 SBN, followed by the Azo-Spiril and Gelati-Vorans. Let me go back to a previous slide. So we selected this guy, the highest peak of the AOA genes in the C13 SBN treatment. We selected this heavy fraction for metagenomic, and we performed metagenomic binning, and we got different metagenomic bins. Fortunately, we obtained the bins associated with the Peltative SB oxidizing bacteria, identifying the SB experiments, that they are Azoacus, Byni, Azo-Spiril, Byn-10, and Gelati-Vorans. And we found all of these three bins hovered the genes involved in SB oxidation, that is the AOA genes. You can see that Azoacus, Azo-Spiril, and Gelati-Vorans all contain the AOA genes. Also, they contain the essential gene for denitrification and carbon fixation. So these genes are the essential genes for anaerobic or naturally dependent anti-monite oxidation. Finally, we get to our conclusion. So the SB contamination or anti-monic contamination substantially influenced the innate microbial community. The microorganisms may use nitrate to oxidize SB3 anti-monite. So bacteria associated with Azoacus, Azo-Spiril, and Gelati-Vorans may be responsible for nitrate-dependent anti-monite oxidation. So these bacteria contain all necessary genes for nitrate-dependent SB3 oxidation. So finally, I would like to introduce, I would like to thank my collaborators, Dr. Xiao and Max and Dr. Fang Bai from our institute. I would also thank, I would like to thank my poll star who is working on this project, Miao Miao Zhang, Ray Xu, and Xiao Xu. So that's the end of my presentation. Thank you. Thank you guys for your patience and time. Hi, Max. Thank you. Thank you very much, Wei Min. Really interesting. And I know there's going to be a lot of discussion on this. I already have a bunch of questions for you as well. But we will get back to that later and continue now with Demetrius Carposa from the Department of Biochemistry and Biotechnology at the University of Thessaly in Greece and examining fungicide applications and how the soil and philosophy of microbial community respond to this. So Demetrius, the floor is yours. Yes. Thank you, Max. I hope you see my screen now. Is that everything okay? Great. Okay. So I would like first to thank the organizers for inviting me to present in this webinar. And I'm going to talk about the response of the soil and philosophy of microbial communities to repeat the application of fungicide. And I'm going to talk about the specific fungicide. So basically, we know that pesticides are applied either as a soiled range or most of them are applied on the upper plant parts, upper ground plant parts. So both plant compartments, the phylosphere and the rhizosphere are expected to be exposed to pesticides. And that means that the microbes that they actually colonize, these two plant-associated compartments are exposed to pesticides and they interact with pesticides on their everyday life actually. So basically, upon these pesticides and counter microbes and microbes and counter pesticides, we expect to have an interaction between these two partners. So the outcome of this interaction can be either toxicity when microbes are exposed to pesticide levels that they cannot transform or use as an energy source or it can be biodegradation when microbes are using pesticides as an energy source. So we can have both the yin side of this interaction and a yin side of this interaction. But if we're looking at the literature, the overwhelming majority of studies looking at these interactions, they have focused on soil. So for example, we've seen both types of outcomes of this interaction in soil and I put some examples here where you can have a growth linked enhanced biodegradation of a pesticide like coxamil which is actually coupled with a gradual increase in the abundance of bacteria that they carry relevant catabolic genes like CEHA. Or you can see a response, the opposite response, you can see a reduction in the rate of nitrification as you see here at the high concentrations rate, concentration rates of certain chemicals like a prodion, for example, when it is applied at times 10 or times 100, its concentration, recommended concentration. But this is all about soil. How about phylo sphere? So we know basically very little about the outcome of this interaction in the phylo sphere. So we actually tested the hypothesis that phylo sphere and rhizosphere exhibit similar responses to the repeated exposure to pesticides. And this, as I mentioned before, the outcome of this interaction could span from accelerated biodegradation when you see microbes rapidly adapting to this pesticide substrate and to grow and proliferate and degrade it rapidly. Or you can see toxicity effects. And to test this hypothesis, we had to select two main components of our experimental plan. First was to select a pesticide. And this was our choice with the prodion. It's an old-fashioned fungicide that is used in several crops. The criterion that we prefer the prodion was the fact that it is relatively biodegradable. But it is also applied as a foliage fungicide, but also as a soil drench. And the second component was to select plants that we include in our study. And we selected pepper as a plant because a prodion is registered for using pepper crops and is registered for used either as a foliage-applied chemical, but also through the drip irrigation system. With these main decisions, the main decisions of our experimentation already made, we set up a pot experiment with 62 pepper plants. We transplanted all these pepper plants at the three to four leaf stage in our pots, and we left them to reach flowering. Flowering is the stage of pepper plants where a prodion application actually commends in practice. So when plants reached flowering, we started applying a prodion. So in 30 of these pots, plants were just treated on the leaves. 22 of these pots received a soil-drenched application of iprodion, and we had 20 pots that they received no iprodion. Instead, they received water either on the leaves or on the soil. And this application was repeated four times at 30-day intervals. And just a quick look in the application and sampling plan. So you can see we indicate with red arrows the iprodion application time points, with the thin green arrows the time points where we extracted DNA from leaves and soil. And with the blue thin arrows, you can see the time points where we took samples for HP analysis to determine iprodion transformation. At the end of these four applications, our application sim of iprodion, we took samples from the rhizosphere and the phylosphere in order to try to isolate iprodion-degrading bacteria. So what did we measure? We actually measured the pesticide transformation on the leaves and the rhizosphere soil. We applied applicant sequencing analysis for prokaryotes and fungi in order to see how microbial community in the two plant-associated compartments respond to iprodion application along this experiment. And at the end of this application sim, we tried to isolate bacteria that could degrade iprodion, both from phylosphere and rhizosphere via enrichment cultures. So let's move to the results. So first question was to see if what we actually... how microbes are responding to iprodion continuous exposure? Do they degrade rapidly or are they struggling with this repeated exposure sim? So it seems that both microbial communities, both in phylosphere and rhizosphere, they seem to adapt to rapid degradation of iprodion. In the upper panel, you can see the results for the soil where you can see right from the first application, iprodion degrades quite rapidly, which was not surprising if you look at the literature, iprodion is relatively biodegradable. So, but still even if we started a very rapid... with a very rapid degradation of iprodion, still it became with repeated application even more rapid. At the fourth application, we had a dt50 of 0.4 days. When we move to phylosphere, you can see that this enhancement of the biodegradation of iprodion was much more prominent. And this is because we had a very slow degradation at the first application and gradually after the second, third and fourth application, we started getting a faster degradation with dt50 at the fourth application of 5.9 days. So in fact, we had an enhancement of the degradation of iprodion in both studied compartments. And then we tried to see how this is mirrored in the composition of the microbial communities in the two planned associated compartments. And we applied some multivariate analysis to determine if the application of iprodion actually changed significantly the microbial communities. And you can see here that... you can see here in the rhizosphere of bacteria and phylocytobacteria, we had some significant changes and iprodion applications significantly structured the microbial community in both planned associated compartments. For archaea, that was also the case in the rhizosphere, but not in the phylo sphere where archaea didn't seem to respond to iprodion application. So when we move to the fungal community in the two compartments, you can see that we had also a significant effect in the composition of the fungal community in both the rhizosphere and the phylo sphere, which was not really unexpected considering that the iprodion is really a fungicide. I'm not going to go into details and tie you up with giving names of a list of genera that they were increased or decreasing abundance, but when we look in the functions that some of these bacteria or fungi are involved, the ones that saw differential abundance in the response to iprodion, we could see that the application of iprodion, both in the phylo sphere and in the rhizosphere of pepper plants, seemed to affect the relative abundance of putative plant pathogens or putative human pathogens of mycoparasites and of bacteria and fungi that they are known to be involved in organic carbon decomposition. But one important and rather interesting observation that got our attention was the fact that the abundance of OT use belonging to Candidatus nitrososphera, which is considered a dominant ammonia oxidizing archaeal lineage in most agricultural soils, seemed to be negatively affected by iprodion application in the rhizosphere. And this is really in agreement with our previous measurements that we've done in a different experimental settings with iprodion, where we also noticed that the main Candidatus nitrososphera were really negatively affected by the application of iprodion. So the next step was to try to isolate iprodion degrading bacteria and we set up some enrichment cultures both from the phylo sphere and the rhizosphere and you could see that in the enrichment culture we see some really fast degradation in both compartments and we isolated eventually three degrading cultures that they were pure and two of them were coming from the rhizosphere and one was epiphytic. And when we actually sequenced their 16S rRNA gene to identify to put them in the phylogenetic context, you see that they were very similar regarding the 16S rRNA gene but not identical and they seem to actually be sister and affiliate with other pynarthrobacter strains that they are involved in the degradation of organic pollutants. But very much it was very interesting to see that they are very, very closely affiliated with other pynarthrobacter strains that they are involved in the degradation of iprodion. In fact, the vast majority of iprodion degraders that we know so far, they belong to this genus, pynarthrobacter, which is really interesting because they have been isolated from Japan, from Greece, from US, all these pynarthrobacter strains, but all degraded iprodion. So we tried to look a bit further and see the transformation pathway of iprodion by these two, by one soil-derived and one phylosphere-derived strain and we noted that they both shared the same metabolic pathway. iprodion was degraded through the production of two intermediate metabolites that they were eventually transformed into three, five dichloroannily. And this is a pathway that is actually also observed in most previous soil, the soil-derived iprodion degrading bacteria. So it seems that we isolated iprodion degraders from two plant-associated compartments that they are phylogenetically very close to themselves and to previous iprodion degraders that all belong to the genus pynarthrobacter, and they all share the same metabolic pathway. So I would say that we hypothesize that maybe there is a particular phenotypic feature of pynarthrobacter that facilitates their specialization for degrading iprodion. So to sum it up, it seems that iprodion repeated application induced responses, similar responses to the phylosphere and the rhizospheric microbial communities. We observed on the one hand enhanced biodegradation of iprodion which came with a mobilization of pynarthrobacter bacteria in both exposed plant-associated compartments. And at the same time, we both observed changes in the composition of the microbial communities in both studied compartments which really appear as affecting microbes that they are essential for the plant soil system homeostasis. So what's next? We would like to look a bit more on the mechanisms that lead to enhanced biodegradation of iprodion in the phylosphere and see how this is actually evolved. Is it coming from soil bacteria that they are mobilized from the plant phylosphere or they are true phylosphere inhabitants that they are actually adapted to iprodion degradation. And also to explore the arsenal of catabolic genes and look at more on the revolution and organization in iprodion degrading bacteria from the phylosphere and the rhizosphere using comparative genomics. There was a recent publication on this area which compared the genomes of two pynarthrobacter strains that they also isolated from Japan and they were they both degraded iprodion. But it seems that the iprodion transformation pathway it's not mature actually genetically. So we would like to enrich a bit more on this. We have now six pynarthrobacter degrading strains and we think that we will with comparative genomics add a bit more information on this. So to conclude I would like to thank the people that contributed and these appear with blue dots in this photograph of the lab and really this work was supported by money coming from two AU funded projects. Thank you very much and I'm looking forward for questions. Thank you very very interesting degradation studies and we'll come back to to questions later and then it's my pleasure to well thank you and then introduce our final speaker for today Karen Boitz from the department of bioscience engineering at the University of Anfra in Belgium and we'll hear about green infrastructure and atmospheric pollution and how this shapes the microbiota. So Karen for is yours. Okay thank you can everybody hear me and see my screen. Yes. Okay thank you. So thank you for the introduction and first of all thank you for giving us the opportunity to present our work on the Phyllosphere Bacterial Communities in Urban Settings. I would like to say that this work is a result of a multi-disciplinary team effort. Both microbiologists and ecologists so I think this is very important and I first would like to thank my colleague Renke. She equally contributed to this work and she played an essential role as the microbiologist in this study. So it leaves me the ecologists to present the work we did. So let me present you with the study we did on Phyllosphere Bacterial Communities in Urban Settings and we looked at the influence green infrastructure in the city has and atmospheric pollution has on the diversity and the composition of these Phyllosphere Bacterial Communities. So as Dimitrios already explained the Phyllosphere it's all the above ground surfaces of a plant on the interface between the plant and the atmosphere and the dominant part is represented by the leaves. It provides a unique but we are rather harsh habitats for microbial life. It's colonized by fungi, archaea and bacteria and these bacteria are the numerically dominant. You can find 10 millions of bacteria per square centimeter of leaf surface. The composition of these communities varies from tree to tree and it depends on the host species and we also see a lot of spatial and temporal variation between these communities. These Phyllosphere Bacterial Communities they influence the health and the fitness of the plant by pathogenic and antipathogenic effects and as such they also influence plant community diversity and the biomass they produce. For example we see disproportionate loss of fitness in some dominant plant species or it can happen that the rare species produces more seeds through a compensatory response to pathogen infection and it's also proven that the influence the ecosystem functioning and the ecosystem services provided. You can think of carbon sequestration, nitrogen cycling or also the mitigation of air pollution by plants and last but not least they also provide an important source of airborne bacteria which of course there are sorts of bacteria to which humans can be exposed. Next to that we also look to urbanization and urbanization can be defined as the expansion of the urban environment and we can see cities as heterogeneous dynamic landscapes with of course high population densities but also changes in land use in land cover biochemical biochemical cycles in climate and hydrology and we also know they have huge demands of energy and water and enormous emissions of carbon. In this urbanization this expansion of the urban environment has effects on global and the local environmental settings and it decreases the local biodiversity and it also accelerates the phenotypic changes in plants and animals. The question we had was okay this urbanization it's a global trend but how does it affect the phylosphere bacterial communities. For this we did not want to work with an urbanization gradient that's how it's done mostly but we wanted to work with a landscape ecological approach so we looked at how intertree variation in diversity and composition of bacterial communities in the phylosphere of trees in these urban settings are related to specific urban disturbance factors and these urban disturbance factors are listed as mechanisms that cause these changes in local biodiversity and these phenotypic changes in plants and animals. We cannot investigate all of them of course but we took out some and the first was the loss and fragmentation of native habitat and the second was the influence of novel disturbances and more specifically we looked at the effect of air pollutants. So we went out in the fields and this is how we did the work so we selected 55 London Plain trees in the city of Antwerp which is located in the north of Belgium in the center of Europe and the trees were in a range of 11 kilometers apart. We selected London Plain because as a host species of the phylosphere bacterial communities because it's rather common tree throughout cities and also in the city we we investigated. It's also really easy to recognize which is kind of convenient when you're working with microbiologists and it's canopy or at least some parts of the canopy are out of reach for people to touch so it decreases the chances of contamination. So the city of Antwerp is the second largest city in Belgium. It houses about half a million of inhabitants on 200 square kilometers. It's this typical European medieval cities like in the center we have this old city center with residential and in commercial areas and that's surrounded by a heavily trafficked ring motorways motorway and the city suffers everyday suffers from huge traffic congestions. Outside the ring road we have these more residential areas with more green areas in between and in the north we have the port of Antwerp which is the second largest in Europe and in the south we have specific precious metals refinery plants and there we find elevated levels of lead arsenic and cadmium in the air. So scattered throughout the city we selected those trees and these trees or the site they grow in. We allotted to five urban categories which were trees in urban green along busy roads and residential areas in industry and harbor areas and along the water edge and from these trees we took leaf samples and they were washed within a buffer solution and here you can see my colleague Wenke at work taking the leaf samples to the lab. There we isolated DNA and we performed sequencing on the V4 region of the city's ribosomal RNA. So let me take you through the main results of this study. So first we looked at how these communities differed between the different urban categories we had in our study. When we looked at the outer richness we saw that surprisingly the richness of these communities was twice as low in the phyllosphere sampled in these urban green sites in comparison with trees along busy roads or in urban residential areas or in industry and harbor areas and then we also calculated the beta diversity of these phyllosphere bacterial communities and then we see that we find higher levels in these urban green sites but this seemed to be caused by both a dispersion effect as a location effect so we could not draw any conclusions from that. On the right you can see a biplot of the PCA we did on the composition of these phyllosphere bacterial communities and you can see with me that we were not really able to see any clear clusters according to the urban categories we analyzed. And then we took a look at the core community. So the core community consisted of 62 OTUs and they were dominated by those typical phyllosphere bacteria, sphingomonas, immunobacter, pantoa, yantino bacteria and sphingomonas, phragi. And we saw that on trees in urban green sites we saw that these core community was really abundant, very much more abundant than at the other sites especially these along busy roads and in residential areas. So that was quite strange to see that this core community was really much more abundant in these urban green areas. So for me these analyzes were not satisfying so we wanted to do some more digging and we wanted to look at these specific disturbance factors in particular. And the first one was with air pollution and for this we did some biomagnetic analysis and this analysis we do biomagnetic analysis of leaves and in that way we measured the saturation isothermal remnant magnetization which is the magnetization that remains after a sample is magnetized in a very strong field. This saturation is a thermal remnant magnetization or short serum we call it, it's a proxymetric for air pollution. So it's shown to relate with particulate matter and trace element, content in particles that are deposited on the leaf and they're a good indicator of time integrated exposure to PM10 but it also relates with nitrogen oxide concentrations in the atmosphere and with particle bound trace elements which are co-emitted or adsorbed to the to these particles. So it mainly relates to exposure to combustion exhaust emissions by motorized traffic but also to emissions from industrial activity. So it is not a direct measure of atmospheric concentrations but it's a time integrated exposure proxy of PM10. And to our surprise we cannot find a relationship between the richness in our communities of the phylosphere and air pollution. And then we looked at the composition and for this we calculated the beta diversity again but this time we did a beta diversity partitioning. So we split up this beta diversity in this resultant dissimilarity and in a Simpson dissimilarity. So the the theness of this resultant relates to shifts due to subsetting of the communities. And the Simpson dissimilarity relates actually to the an idea gives an idea about replacement of species by other species within a community. So that's what we've done and you can see this in the in the graph. So the left in the center of the slide you can see theness of this resultant beta diversity and it's plotted against the dissimilarity in air pollution proxy. And on the right you can see the turnover beta diversity which is plotted against the dissimilarity in air pollution exposure again. And we saw that there was no relation with theness of this resultant beta diversity. But we did see a significant positive relationship between this turnover beta diversity component with the difference in in air pollution. So and then we looked at the most abundant general and then we couldn't really see a lot of obvious things and a lot of changes. The only thing we saw was that the abundance or the relative abundance of rostone was increased when the level of air pollution was increased. So based on this we made the conclusion that you can see shifts in community composition occurring in these bacterial communities in the form of taxa turnover in the form of taxa turnover as related to air pollution. So in this case we can see species disappear and that they are replaced by other species that appear. So this suggests that exposure to the air pollution relates with changes in the composition but not in richness. So it's really a balanced equilibrium of taxa gain and taxa loss and in the end in net there's no loss of taxa observed. So for this we hypothesize that air pollution can affect the phallus or bacteria either directly as because air pollution can be a resource or it can be a stressor. But it can also be indirectly through effects through the plants via changes in leaf characteristics. Like we've seen effects of air pollution on stomatal density and leaf petability and the specific leaf area for example in many of our previous studies. And then we had a look at another urban disturbance factor which is change in land use, land cover. And for this we looked at the urban atlas. The urban atlas is a layer of land cover and land use which is produced by in the Copernicus land monitoring service of the European Union and the European Environment Agency and it provides information on land use and land cover at a 2.5 meter resolution. And from these maps we calculated several landscape metrics like land cover richness and diversity. Those are land cover proportions of the different land cover land use types we can find in this map. Among which were urban fabric, water, roads but also green infrastructure which we defined as everything that's urban green which is agricultural and semi-natural are also including forests. If you ever can find them of course in these densely built up cities. And then from these landscape metrics we saw that there was a relationship with the alpha diversity in our communities. So we saw that when the proportion of green infrastructure in a buffer area around the tree increased and we saw that the auto-originus decreased. So the number of auto-use in our communities were reduced when we had less green in the surroundings of our tree which was quite strange though and unexpected. Then when we look at the composition again we did the beta diversity partitioning and here we didn't really see a relationship with the turnover component of beta diversity like we've seen for air pollution but this time we saw a relationship with the nested nest resultant beta diversity. So with an increasing difference in the proportion of green infrastructure in a buffer zone around the trees we saw that there was an increase in the nested nest resultant's beta diversity. So the more trees differed in the amount of green infrastructure around them the more they moved away from each other in composition of phylocea bacteria on them and this was related to nestedness. So next to that we also looked to the most abundant species and we saw some species like really increasing in abundance in these areas with a lot of green infrastructure like hymenobacteric finger monosembaring here like this really typical phylocea bacterium. While others seven genera in total they decreased like acinetobacterus caramonella those are generally decreased when there was more green infrastructure surrounding the tree. And then the quark community then we saw that it really increased in relative abundance when there was more green. So when we put all of this data together when then we can see that the bacterial communities on trees in areas with more green infrastructure there really can be seen as a subset of the communities that occur in areas with less green. And so we see that the presence of more and also closer to green infrastructure to a tree reduces its diversity. And that's really strange because that's opposite to what actually is observed for in urban areas for a lot of taxa like thinking of insects and birds where we see that when there's less green infrastructure available we see that the diversity the number of species declines. So this was contrary to what we expected actually. And then when looking at the community composition we see that that in these green when there's a lot of green infrastructure we see this reduction in in in diversity but also it's like the quark community gets stronger and stronger. So plenty of genera were introduced in the community when there was more of the other land use or land cover types available and those were mainly anthropogenic land use and land covers. And the species with higher abundances in the phyllospheres trees in the green areas could be seen as like the more autochthonous plain phyllospheres specific quark species and they're more adapted to resisting these harsh environments. But next to that there are taxa that were introduced and increased in these areas that were more anthropogenic with less green and they may originate from typical urban sources like concrete or just people passing by all the movements of the cars like with all the the dusts that is re-suspended in the year. So the bacteria coming from these non-green land cover types could be they enrich the urban phyllosphere but they could be considered as more the exotic species that come and enrich these phyllospheres bacterial communities. It could be that this is related to lower dispersal in these areas with more green infrastructure but it could also be related to ultra selection pressure of course. So this the concluding slide is that in these in these city areas we see that there's an effect of green infrastructure which leads to lower diversity but the presence of more core auto use that more reduce typical phyllospheres species while the effect of air pollution is more related to turnover so some species disappear other species appear and in the end we have the same diversity. So that's the story we we had in this in this paper. If you want to read more about it then I would say go read our paper but any questions are welcome. Thank you. Okay thank you Karen and previously thank you again Dimitrios and Waymin and Stefan. I think we have time for discussion and any questions that are coming from the audience so again please send in your questions through the Q&A function in a go-to webinar and we will then respond to them but let's while I go through what we have coming in maybe Stefan you want to to start I know you had some aspects that you wanted to get into. So maybe we start with somebody who's really late in the day I don't know how late it is Waymin at your end but maybe I can start with you. I think it's already tomorrow Waymin so thank you for staying up. Okay so the question for tomorrow and yes so it was really interesting I mean you showed lots of stuff that was also not in the paper but I guess one common thread between what you showed and what's in the paper is about the when you get really these these different patterns of metabolism in particular those related to to antimony and I was wondering if you could see a clear pattern in your studies with redox potential in different areas of soil you will have different redox potential so did you see things coming up okay relation to redox potential and maybe soil depth. Yeah that's a good question so actually the reviewer also asked the same questions so what is the redox potentials of the death reserve soil profiles unfortunately when we're doing the sampling we don't have instrument to measure the redox potential I think it's kind of difficult to measure the redox potential in situ so my answer is we don't I think we don't measure the EH or any other parameters related to the redox potential yeah but this is a good question actually yes but maybe from the names of the OTUs that you got you can infer maybe something which is more anaerobic or more oxy yeah actually I think maybe the more anaerobic is well you know enriched in the bottom soil and the surface soil is kind anaerobic so I think this but I didn't get a chance to look back to my papers so we published this paper last year so I may go back to see what happened in the in the soil profiles so this is one aspect I mean you focused of course on the antimony either reduction oxidation and that's of course for the microbes with this metabolic capability a potential energy resource and so you would expect that they would become enriched what about other key ecosystem processes I mean what I mean so you have contamination through antimony how is that affecting nitrogen sulfur carbon cycling and what are so called the broader ecological consequences yeah actually this is a good question so so in this case we mainly focus on the antimony cycling because we focus on the microorganisms microorganisms persist participating in this element this antimony cycling however when we go back to tailings the tailings is oligotrophic environments so in that case we found a very specific biogeochemical process relating to the antimony cycling so we found the sulfur oxidized antimony reduction so in that case the microorganism the microorganisms didn't use the organic matters as the carbon source they used the inorganic carbons I think this is the a case for the capability of the microorganisms thriving in some oligotrophic environments so in this in in the oligotrophic environment they developed or they involved some very specific uh metabolic trains or metabolic potentials I think this is a case showing how the microorganisms linking the carbon the sulfur and metal cyclings so I have a question for Dimitrios the the strains you isolated uh that degrade um I so I think you said or wrote that they are actually not visible in your in your diversity profiles right so so they are very low abundance uh did you try to check what what is the actual abundance using another technique then then then uh you know sequencing did you try to do qpcr something do you have an idea how rare these these organisms are yeah um yes indeed uh we when we looked back in the pinearthrobacter that we've isolated and we looked back in our data set to see if these were actually somehow enriched during this application process we found some ot use some pinearthrobacter ot use that they were not actually the ones that we isolated so it seems that they were very low at very low abundance these pinearthrobacter that we isolated but yes we didn't actually go back to uh follow up their abundance during this enrichment process and this is um always um I would say a problem that might arise when you use enrichment cultures for isolation somehow you might um you know you might select something that was not the main player in in in situ in the phylosphere or in soil or it was a very slow degrade a very slow grower which it's actually a pinearthrobacter are very slow growers and very lazy uh microbes in soil at least and uh if you give them in enrichment culture the right the right uh favorable conditions you might uh see them uh to to grow fast and you isolate them or of course during the enrichment culture you might isolate uh something that actually picked up the function during the enrichment culture okay but it's strange that uh pinearthrobacter seem to appear always regardless of the environmental compartment you are looking they appear always as a iprodion degraders which is um I'm not a great fan of the of the specificity of bacteria when you consider that uh most of these catabolic pathways they are uh driven by uh plasmids and by mobile genetic elements uh but uh here it seems to be uh somehow some sort of specificity in this capacity this phenotype uh specific uh relation between pinearthrobacter and in prodion transformation and we would like to actually look at this a bit further with uh genomic analysis to see what actually triggers this uh specialization but uh we didn't actually trace back with qPCR of these pinearthrobacter strains that we isolated so you're seeing fairly rapid degradation on their end so this is one question here from the audience so if the fungicide is degraded so quickly is it still effective well that's a good question because uh when we talk about enhanced biodegradation we actually uh in a lot of cases you lose efficiency so that's another question that it was raised by uh by our paper by our the work that this enhanced biodegradation that we look with repeated application so enhanced biodegradation was always the case and we knew about it in soil iprodion was one of these pesticides that in soil suffers from enhanced biodegradation and it loses its efficiency uh but um in philosphere it's the first report of this phenomenon of enhanced biodegradation and if you consider that iprodion is also a fully as applied fungicide uh you often uh i mean you you could expect that it has a reduced efficiency and this aspect has been neglected a lot in uh for insect insects control and fun fungi control that most of the times we link it to uh resistant mechanisms but uh biodegradation could be also a mechanism that leads to uh reduced efficiency so here's one question thank you for uh Karen and uh the where do i find it oh in terms of the radius i mean so you looked at the green space around uh so what radius around a plant would you consider important as a bacterial source to take into account and i was wondering a follow-up to that question what about particulate matter what's the microbial community on the pollution the particulates that are landing on the leaf and could this be a source of why there is so high diversity so this question of what area around a plant but also what would be other sources yeah so we i we tested different uh distances for these buffers so we started with buffer zones of 20 meters and we went back went all the way to one kilometers if you go further than that then you cover almost half of the of the city so that's no no point there um but then we look and then we saw for all the analysis we did we came actually to the same conclusion that that's distance of 100 200 meters we had the best fits or or sometimes it was the only fit uh good fit we we got so it seems that's a distance of 100 200 meters is a is a range in which this green infrastructure has a as a significant effect so i i think there's also been a study that looked then into these the composition of airborne um of these airborne bacteria in the in the atmosphere and they also found in even within the city uh differences in composition big differences in composition um at different locations in the city so it really has a high variation in these uh and the sources of of um of bacteria which can land in the in the philosphere of course yeah so based on our data it seems that 100 200 meters is a good range and then regarding the the bacteria on the p.m that's um i don't know much about that but um if you look at the formation of your primary p.m um so primary p.m coming straight from from the source and and next to that you have the secondary p.m which is formed in the atmosphere um so this the primary p.m if it's uh it comes from the exhaust pipe from from vehicles it's it's quite it's it's very hot so i don't think that a lot of bacteria will reside on the surface of these particles um but of course the p.m that comes from the resuspension of of dust yeah that can be loaded with with soil part with soil bacteria of course yeah and then the the secondary p.m which is formed in the atmosphere they uh they're loaded yeah with these airborne bacteria yeah um and of course there's um an interplay between the bacteria that are in the atmosphere in which you are on the leaves um because the leaves are continuous source also bacteria in the in the surroundings but then the bacteria that are on the leaves also depend on what's coming in through this parcel from from the atmosphere so it's difficult to say here what's the source what's the what's the sink uh they're in continuous interaction okay thank you yeah there's one sort of overreaching question for everyone this is from Tillman Looters um on the aspect of ecotoxicology with higher organisms that is always coming up with essentially predicted no effect concentrations or ld 50s or or something like that is there's going to be a corresponding microbial indicator whether that's in uh mining sites or the urban environment or agricultural systems with uh use of pesticides so is there something that will be some microbial indicator that we can use uh broadly to understand what are the consequences to actually anyone please jump in i i can start if you want um well i could i could certainly um um answer that for the agricultural environment so from the point of view of of microbial functioning i would say from my experience and from what that comes up in the literature in the last five or six years um we've actually come come to a conclusion i would say that there are two main microbial groups that they are more responsive to pesticides or organic pollutants i would say or perturbations in soil environment and these are the ammoni oxidizing microbes i would say first and then it's arbuscular mycorrhizal fungi which always with symbiotes obligator symbiotes that are like arbuscular mycorrhizal fungi is sometimes difficult to tell um to separate this toxicity to plant which is the host or the microbe itself but uh ammoni oxidizers are really good indicators because they are seem to be ecotoxicologically responsive to pesticides we have good tools to measure the diversity to measure their function um and they are very important organisms for um and cycling in in soil so i would say that i would actually put forward the first ammoni oxidizers and then arbuscular mycorrhizal fungi as two potential indicators of uh toxicity induced by pesticide application okay thanks women uh the metal contaminated sites what do you think is going to be some kind of a measure to understand how problematic or polluted a site this to sort of have a dipstick to say yep this is what we need to do so actually i i don't think which bacteria can be the bio indicators but in our cases we found many bacteria are very capable to transform the the metal so i think maybe they can be indicators for the for the potential for future bioremediation for example the geobacter we found the geobacter are responsible for anti-monad reduction in many in many rice paddies we performed the DNA seep and we found we took the soil samples from across the southwest China from five different five different uh five different regions and all of them so we performed DNA seep and we found all of our geobacter was identifying as the anti-monad reducers in all this site in another case we found thalobacillus the thalobacillus are very capable in the tailings so this guy can do a lot of job for example they can oxidize the arsenide they can also reduce the arsenate they can oxidize the anti-monad and they can reduce the some and others other metals in the in the tailings so i i don't think there might be a universal bio indicators in the mining area but if we can focus on the soil types for example the rice paddies or the agriculture soil or the tailings maybe we can we can point out we can pinpoint some very super bad maybe super bad or maybe very capable bacteria so that's my answer okay and karen what do you think about the the urban environment yeah difficult question but it's a good one um but it's difficult um we see so many so many um taxa there but it's one thing is clear for like um but maybe that's not that interesting but an indicator for more the more available green in the surroundings and we could say it's clearly heminebacter in finger monas it's that's clear from several of our studies that those are the most thriving in these more greener areas regarding air pollution it's still a question we're working on but we see for example uh we see us kermanela and rastonia popping up as um as species that are that seem to be able to cope with these high levels of relatively high levels of air pollution um but to pinpoint it to to specific taxa it's i think it's really too early to do that because a number of studies working on this on the effect of air pollution on these full-sphere bacterial communities are it's um it's still limited but uh we're working on it okay thank you so stefan any additional question here or add to that i'm not an expert on these studies of course but reading through these papers i think for tilma not the rudors also i guess they may be an extra twist to this to this minimal concentration because from dimitriosis and karen's paper i i became aware of the fact that the substance which is toxic to a range of organisms can actually uh by mitigating their development give rise to other bacteria which are then pathogens so it's would be an indirect effect which maybe could also be measured which is interesting like you had a in dimitriosis case you had the pesticide and then the fungal pathogens disappear but the human pathogens may come up and i guess karen has seen similar things happening in the urban system or at least it was alluded to in the paper so indirect effects with pathogens human pathogens coming up that could be also an interesting measure of you know minimal concentrations of toxics that should not be increased yeah yeah these indirect effects i think they can be very important because we're always thinking in in a way of direct effects but the interaction with the plant is is very important in in our study with in the philosophy and it is very plausible that the effects we see on the phylosphere are are indirect and in that way have an influence on the composition of the phylosphere like for example we've seen the effects of air pollution on only like only morphology only we can see that there's an effect on stomatal density which of course has an effect on where where bacteria can reside on the leaf the specific leaf air the dca so the the leaf wettability of it's also influenced by air pollution and it's known that it relates to the with with the bacteria that occur on the leaf so yeah the indirect effects are very worthwhile um yeah looking into and i think the other aspect to remember is that even though the community might drastically change the functions might still be there because of all of the redundancy so going back to how is that affecting core ecosystem services even if the players are completely different so no it's a different difficult question so let's see i think we're going to have to wrap up i mean we're now into an hour and a half so Stefan any final questions or comments or i think this was really as as we expected a real diverse overview of different aspects of microbial ecotoxicology as an expression as a term for those who were wondering what it meant i guess we know a little bit more now yeah it's a little bit of everything so thank you there are still a few questions left that we didn't have time for so we will distribute these by email and you may then get a response just for you in the audience to know that there are still things that we'll try to get back to you with specific answers so again no thank you Karen, William and Dimitriou, Stefan and everybody in the audience this has been a really interesting session and hope to see you again in about a month when we have a webinar on microbes and metals so they'll continue on the theme that Wayman had and there's another FEMS webinar next week on vaccines which of course is on everybody's mind right now and while we one reason why we are are doing these webinars as well so thank you again and looking forward to seeing you all in person soon again thank you