 production and I'll get to it without any further ado. So just a little bit of background, a lot of people here come from obviously fine pathogen background but there's an estimation that around 20 to 30 percent of the global crop production is lost to pest and pathogens and it's largely due to fungal pathogens and as Rebecca said our group sort of investigated and specializes in investigation of fungal pathogens. In chickpea one of the major folio pathogens is a rabiae. Edo, sorry to interrupt. I'm not able to see your screen. Is everybody else able to see your presentation? Just type in the... It's looking fine for me. Yeah, it's looking okay. Oh, okay, that's good. That's okay. All right, sorry about this Edo, continue. Okay. So yeah, I was saying in chickpea as coca either rabiae is one of the major fungal pathogens and again on optimal conditions it can cause up to 70 percent or even 100 percent loss of yield. Why work for chickpea industry? Well, it's a very it's a major industry in Australia. The production has grown sort of gradually over the years started originally from the more temperate southern regions of South Australia and Victoria but then really expanding into New South Wales in the 2000s and then into Queensland as well and actually and again for this it is and as you can see in 2017 there was a huge expansion into Queensland more than doubling the production. So it's a really major crop system in Queensland so there's a lot of effort. So worth noting that after that big year of 2017 the Indians markets imposed an import tariff which pretty much dropped the production because it wasn't worthwhile anymore and the production went to what it was like 15 years ago 12-15 years ago but we can see that now it's the tariff is easing and the production is picking up again in New South Wales and Queensland. Again the production of chickpea in Australia it's a temperate legume so it is more in the southern side of Australia along the east coast and it goes all the way north to central Queensland around Emerald and then down along New South Wales, Victoria and South Australia and there's a little bit of production as well in western Australia over at the west. There's a very tiny area of pocket of production in the northern territories in the Orden River but they have been safe from the pathogen so far. Worth mentioning with those different regions that there's different climates obviously different soil types and then also different effect on the pathogens. So for instance the northern regions they're less affected by ascokite arabia so they use more susceptible cultivars whereas the southern regions where the industry has been established for a lot longer they really suffer from ascokite arabia in some years so they try to use more resistant or moderately resistant cultivars. Ascokite arabiae it's an ecotrophic ascomycite fungus that causes ascokite blight. It exhibits a range of isolates that differ in their morphology and their pathogenicity so it ranges from non-aggressive at all to highly aggressive and basically killing the plant. It is a haploid with only a single mating type observed in Australia all right it's a very important point that at the moment as far as we know it only reproduces clonally in Australia. There is a reference genome available that's been constructed by Curtin University the CCDM and it's pretty good quality and they keep approving it so we do have a fair bit of genomic resources to work with. So our work our group has been focusing on monitoring us quite a rabiae in Australia and in terms of the entire sort of view of the pathogen of the disease we focus on the pathogen. So there has been a couple of GRDC funded projects one of them running from 2013 to 2018 and then another one that is currently ongoing until 2024 with the following aims. We aim to monitor and characterize isolates that have been collected from chickpea paddocks and basically maintain the isolated collection of Australia the national isolate collection for us coca either rabiae. We then assess and characterize them and then we deliver the worst or most highly aggressive isolates back to the breeders to be used in the selection and then we want we also try to identify the dispersal the adaptation and the evolution patterns in the population so we can understand better how those highly aggressive isolates emerge and under which conditions. Then we want to dive deeper into the genome of those isolates to identify specific virulent genes, effective molecules or any other genomic variants that may contribute to those highly pathogenic variants. Using those those tools we hope then to develop more applied molecular assays that allows us to rapidly pathotype in the field or to be able to determine whether isolate is highly there's a highly likely to be of a great risk even before we run through the bio essence. So far we've collected more than 3000 isolates from 2013 until now and it's been a major effort by state plant pathologists, agronomists, breeders and growers that have been helping us with the collection. We get them to Griffith, we single-sport them and then we store them and as I say we also characterize them. These isolates represent the variety from the different regions, host genotypes, aggressiveness and over the years as well. Each year we choose we're choosing a subset of isolates somewhere between 100 and 200 and then we actually assess them. We're running bio assays on a differential set of hosts to determine the pathogenicity levels and then we also genotype them and I'll show which methods we've used for the genotyping and we also assess the mating type using the PCR. The pathogenicity assessment is done against the differential hosts that range from more resistant cultivars or breeding materials such as ICC 396 and Genesis 90 and CIMR towards the most susceptible ones such as BBA-Hitrake and Kiabra which is a very susceptible host which is basically our positive check. We can see here the list and how we classify them based on their response to those cultivars and we can see that we pretty much classify them from pathogenicity group 0 to 5 where 5 is the most highly aggressive and 0 ones that are really non-aggressive only cause disease symptoms on Kiabra and not on all the other cultivars. We then summarize this information and what we're doing we're looking then how the population or how the aggressiveness evolves over the years and what we found is quite interesting and something I guess all plant pathologists sort of observe and this is a challenge that the industry need to tackle that as the years progress the frequency of highly aggressive isolates increase and these are evidence in those the more hot colors orange and red whereas the green and the blue represent the fraction of the isolates that classify as pathogenicity group as the low pathogenicity groups and we can see that there is definitely an increase there and that emergence of the isolate the pathogenicity group 5 only emerged in 2017 and we see we saw it again in 2020. 2019 was a fairly mild season for the disease which was very good for the growers but also it correlates with the intensity or the scale of production if you notice from the graph that I put of the of the chickpea production in those years 2018-2019 we had a major deep so there wasn't much chickpea grown so this is part of it. Another thing we'll look at is that each of those host genotypes that we assess how what is the percentage of highly aggressive isolates and we can see that there's some dramatic increases you know over one year from 12% to 48% on PBA hatric which is now considered as a moderately susceptible cultivar and then as well some of the more resistant cultivar such as genesis there's a gradual breakdown of its resistance and we saw PBA cimer that was released not very not too long ago and it was in our test it was fairly resistant until 2018 and then within pretty much two years it completely broke down and now we've got almost 50% of isolates are considered highly aggressive on that cultivar so there's no doubt here that the the pathogen is able to adapt and is able to overcome our resistant sources so as I mentioned part of the work that we do is we're trying to genetically characterize those isolates to be able to infer the population structure and to be able to identify some patterns in their evolution so we started off by doing microsatellite uh genotyping we have seven microsatellite and that was uh markers and that was the work of Yasir Mahmood finished his PhD and he ran those on he ran the he did the genotyping for almost 600 isolates that were collecting between 2013 and 2017 and we found very low diversity in the population very low genetic genetic diversity and in fact the majority of our isolates almost 70% of them all match the single dominant haplotype and still all of them were only one mating type so as we can see here in this network uh image we can see all these comprised of uh just a single haplotype regardless of where the samples came from all right so it it gave us an indication that is highly clonal but there were some question marks there whether the resolution of markers was good enough so we moved on in the following year and we tried to do a similar analysis using genotyping by the sequencing uh in this case using dartsic so we used 200 around 280 isolates and included two middle eastern isolates from the icarta collection and we ended up with almost 1500 polymorphic SNP which was very encouraging we said all right without 1500 SNPs we'll be able to to have really good resolution and to distinguish between the isolates so we did a pca analysis a principal component analysis of all those isolates and what we found very interestingly is that again there is very very low variability so basically the two middle eastern isolates had a fair bit of variance between them but then all the Australian isolates all the 280 just pretty much overlapped on top of each other so in comparison to the other isolates they're all pretty much one clone and the number of SNPs that were polymorphic within the Australian population dropped to only 229 so again there's not a lot of diversity there's um in the population when we look at the third and fourth components of the principal the third and fourth principal components we started to see some uh differentiation between those isolates and we can see that roughly it went by the origin of the isolates so there was a group there's still a large group of very very closely related isolates but then some groups from New South Wales some groups from South Australia and then the border of South Australia Victoria and New South Wales so there's definitely some element of the origin of where the isolates come from that determine their their genetic background we analyzed their their population genetics matrices and again we show we've seen very very low heterozygosity with all the heterozygosity less than 0.02 or close to 0.02 so very very low hetero low gene diversity we have identified that Western Australia shows very low diversity we pretty much half of the isolates all can be can be considered as clones whereas in Queensland sorry whereas in clean in Queensland there's a bit more diversity and there's more genotypic diversity found in Queensland so basically all the isolates each one of them were considered as a clone that stands by its own we then looked at any patterns for gene flow and we used a minimum spanning network on those similarity distance metrics and we found some interesting patterns we found some evidence like I've shown in the principle component before of isolates that evolve locally within the region so within New South Wales within South Australia within Victoria but then there's a whole lot of isolates that are very close to each other but then found in all regions right so that that gives us a hint that those isolate must have moved between one region to another I'll skip that one for because we don't have a bit of time but just very very briefly we found a few genetic cluster that are really highly associated with highly aggressive isolates versus non-aggressive versus clusters of non-aggressive isolates so just to summarize a little bit what the main findings of what we found we found that until now all the isolates that we assessed were of only a single mating type which means that they reproduce as sexual and we know that there's very low genetic diversity so if you put those together but on the other hand we identify very high level or increase in pathogenicity so there's obviously the pathogen is able to adapt now if you look in the literature the evidence says or at least what the literature claim is that these two conditions they should contribute to very slow evolution or very slow adaptation rate compared to isolates that have high genetic diversity and that can reproduce sexually so again it's hard to tell whether we would have seen a much more increased rate when we had the other mating type and luckily we don't have it in Australia but something doesn't match here all right and the question is why why we see that evolution and that adaptation given those sort of very bad starting points for the isolates for the population so we need to we're looking at and we're thinking about what drivers of of what drives the evolution of the pathogen so first of all it can be that modes of adaptation that we've seen that those locally adapted isolates sort of able to adapt better to their own conditions and the hosts that are used there but also those introductions and again because those isolates don't reproduce sexually they don't reproduce spores see what they take that are airborne so it's very unlikely that spores have traveled naturally between Western Australia and Queensland for instance so it must be introduced by human activities such as transferring contaminated seed or machinery other modes of variation that we need to look into is whether we're not looking at the right type of genetic variation we've been looking at SNPs but maybe there are significant structural variations insertions or deletions maybe there are copy number variations maybe there's even horizontal gene transfer has been suggested before that contribute to so so maybe there is a genetic variability but we don't see it in the SNPs another option is that there's epigenetic variation that will change how those isolate behave even though the genetic background is sort of similar then obviously there's the interactions with the host and interaction with the environment that they can contribute to how those isolates adapt and evolve so just what are we going to do next we're trying to use high resolution genotyping so we're moving into whole genome sequencing that gives us sort of an increase in order of magnitude increase in the number of SNPs that we can identify and we can also then work on other types of polymorphism such as structural variants we want to go through some controlled selection or evolutionary studies in the paddock or in trial site to better understand how unknown starting population evolve and as I said whether there's other modes of genetic transfer that we're not aware of such as horizontal gene transfer or maybe our structural variants that we might be missing I just wanted to thank the team well thank you for listening and give me the opportunity to present and I want to really thank the team Rebecca's group the Sustainable Food Production with Rebecca Maldicristi our pathologist and Sam and myself and Niloufar for doing wonderful work assisting us with the population genetics analysis from USQ and now moving to Union of Melbourne Adam Sparks looking at modeling those environmental and soil effects into the into data and obviously the GRDC for funding these projects and the state pathologists and growers that help us collect the samples especially during COVID if you want to look at sort of the summary of this of these results there's the presentation here and just lastly if you have a minute while you go through the question I might just leave that one in if you want you can use this QR code and have a look at the dashboard that we've developed to monitor the isolate that we collect and to provide these summary graphs and tables of where the isolates emerge and so on thank you very much